Immunoreactive glycoprotein gp19 ehrlichia canis

ABSTRACT

The present invention concerns gp19 immunoreactive compositions for  E. canis  and compositions related thereto, including vaccines, antibodies, polypeptides, peptides, and polynucleotides. In particular, epitopes for  E. canis  gp19 are disclosed.

The present invention was made at least in part by funds from theNational Institutes of Health grants R01 AI 071145-01 and 1 P41RR018502-01. The United States Government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention concerns at least the fields of molecular biology,cell biology, pathology, and medicine, including veterinary medicine. Inspecific aspects, the present invention concerns immunoreactive gp19compositions in E. canis.

BACKGROUND OF THE INVENTION

Ehrlichia canis is a tick-transmitted obligately intracellular bacteriumthat causes moderate-to-severe and sometimes fatal disease in wild anddomestic canids. The genomes of E. canis and other organisms in thegenus, including E. chaffeensis and E. ruminantium, exhibit a highdegree of genomic synteny, paralogous protein families, a largeproportion of proteins with transmembrane helices and/or signalsequences, and a unique serine-threonine bias associated with potentialfor O-glycosylation and phosphorylation, and have tandem repeats andankyrin domains in proteins associated with host-pathogen interactions(Collins et al., 2005; Hotopp et al., 2006; Frutos et al., 2006;Mavromatis et al., 2006). A small subset of the more than 900 proteinsencoded by each of these genomes are recognized by antibody (Doyle etal., 2006; McBride et al., 2003; McBride et al., 2000; Sumner et al.,2000). Several of the major immunoreactive proteins identified andmolecularly characterized are serine-rich glycoproteins that aresecreted. Many of these glycoproteins have tandem repeats; however, onehas numerous eukaryote-like ankyrin domains (Doyle et al., 2006; McBrideet al., 2003; McBride et al., 2000; Nethery et al., 2005; Singu et al.,2005; Yu et al., 2000).

Numerous proteins have been identified in E. canis (n=12) and E.ruminantium (n=31) that contain tandem repeats. Notably, threeimmunoreactive proteins with tandem repeats have been identified andmolecularly characterized in E. chaffeensis (gp120, gp47, and VLPT) aswell as two orthologs in E. canis (gp140 and gp36, respectively). Theortholog of E. chaffeensis vlpt gene has not been identified in E.canis, and it has been reported that this gene is not present in otherehrlichial genomes (Hotopp et al., 2006). Extensive variability in thenumber and/or sequence of tandem repeats in the E. chaffeensisimmunoreactive proteins (gp120, gp47 and VLPT) as well as E. canis gp36is well documented (Chen et al., 1997; Doyle et al., 2006; Sumner etal., 1999). The presence of tandem repeats in both coding and noncodingregions of the genome has been linked to an active process of expansionand reduction of ehrlichial genomes (Frutos et al., 2006) and isconsidered a major source of genomic change and instability (Bzymek andLovett, 2001).

Although the E. chaffeensis VLPT is immunoreactive, little is knownregarding its cellular location, function and role in development ofprotective immunity. The E. chaffeensis vlpt gene exhibits variations inthe number of 90-bp tandem repeats (3 to 5) and has been utilized as amolecular diagnostic target and for differentiation of isolates (Sumneret al., 1999; Yabsley et al., 2003). The VLPT of E. chaffeensis Arkansasis a 198 amino acid protein that has four repeats (30 amino acids) andhas a molecular mass approximately double that predicted by its aminoacid sequence (Sumner et al., 1999). E. chaffeensis VLPT protein appearsto have posttranslational modification consistent with other describedehrlichial glycoproteins, but the presence of carbohydrate on VLPT hasnot been demonstrated.

The present invention fulfills a need in the art by providing novelmethods and compositions concerning erhlichial infections in mammals,and in particular provides methods and compositions in an E. canisortholog of E. chaffeensis VLPT.

SUMMARY OF THE INVENTION

Canine monocytic ehrlichiosis is a globally-distributed tick-bornedisease caused by the obligate intracellular bacterium E. canis and is auseful model for understanding immune and pathogenic mechanisms of E.chaffeensis, the causative agent of human monocytotropic ehrlichiosis.In general, the present invention concerns ehrlichial immunogeniccompositions, including, for example, immunoprotective antigens asvaccines for ehrlichial diseases, such as subunit vaccines, for example.The immunogenic composition may be employed for any mammal, including,for example, humans, dogs, cats, horses, pigs, goats, or sheep.

In certain aspects of the invention, there is identification andcharacterization of highly conserved 19 kDa major immunoreactiveglycoprotein (gp19) in E. canis, the ortholog of the E. chaffeensisVLPT. The E. canis gp19 lacks tandem repeats present in VLPT of E.chaffeensis, but the two proteins exhibit substantial amino acidsimilarity (59%) in a cysteine/tyrosine-rich carboxyl-terminal region,and both genes have the same relative chromosomal location. Carbohydratewas detected on the recombinant gp19 and a single major antibody epitopewas mapped to a serine/threonine/glutamate (STE)-rich patch. Thisepitope was sensitive to periodate treatment, and an exemplaryrecombinant protein was substantially more immunoreactive than anexemplary synthetic peptide, demonstrating a role for carbohydrate as animmunodeterminant, in certain embodiments of the invention. The gp19 wasfound on both reticulate and dense cored cells and was also present inthe extracellular matrix and associated with the morula membrane,indicating that the protein is secreted.

In specific aspects of the present invention, there are ehrlichial gp19polypeptide compositions (or polynucleotide compositions that encode allor part of them) with one or more of the following characteristics: 1)comprises one or more carbohydrate moities, which in specificembodiments comprises part of an epitope determinant; 2) comprises oneor more moieties, such as an epitope, that are immunogenicallyspecies-specific; 3) is released extracellularly, such as by secretion;4) comprises major B cell epitopes; 5) is surface-exposed; 6) isassociated with the infectious dense-cored forms of Ehrlichiae, such ason the surface, for example; and 7) is associated with morula membranes(Ehrlichiae organisms form microcolonies inside cellular vacuoles(morulae) that harbor many individual Ehrlichiae) comprising dense-coredforms. In further aspects, recombinant polypeptide compositions of thepresent invention are able to be glycosylated in a cell to which it isnot native, such as an E. coli cell, for example. The recombinantpolypeptide may then be employed as an immunogenic composition,including, for example, a vaccine.

In particular embodiments of the invention, there are E. canis gp19immunogenic compositions that comprise an amino acid sequence that isimmunogenic, and in further particular embodiments, the immunogenicityis characterized by being at least part of an epitope. In furtherembodiments, the amino acid sequence comprises at least part of avaccine composition against an ehrlichial organism, such as E. canis. Inspecific embodiments, the amino acid sequence comprises serines,threonines, or, optionally, alanine, proline, valine, and/or glutamicacid; in additional embodiments, the amino acid sequence isglycosylated. In further specific embodiments, the amino acid sequencecomprises part or all of the following exemplary sequence:HFTGPTSFEVNLSEEEKMELQEVS (SEQ ID NO:13). In certain embodiments, theepitope comprises additional amino acids on the C-terminus, such asthose that are immediately C-terminal to the sequence of SEQ ID NO:13 inthe naturally-occurring gp19, such as is exemplified by SEQ ID NO:17 orSEQ ID NO:19. In particular embodiments, there may be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more additional amino acids on the C-terminus of SEQ IDNO:13. In additional embodiments, the amino acid sequence is comprisedin a pharmaceutically acceptable excipient, which in some aspects of theinvention comprises an adjuvant. In certain aspects of the invention,there is a polynucleotide comprising SEQ ID NO:20(CATTTTACTGGTCCTACTAGTTTTGAAGTTAATCTTTCTGAAGAAGAAAAAATGGAGTTACAAGAAGTATCT) that encodes the peptide sequence of SEQ ID NO:13.

E. canis sequences may be identified following sequencing of gp19 inother strains; additional E. canis strains are tested, including NorthCarolina (Jake), Oklahoma, North Carolina (Demon), North Carolina (DJ),North Carolina (Fuzzy), Louisiana, Florida, Sao Paulo, Cameroon,Israeli, and Mexico. In additional embodiments, the amino acid sequenceis comprised in a pharmaceutically acceptable excipient, which in someaspects of the invention comprises an adjuvant.

In certain embodiments of the present invention, there are immunogenicgp19 E. canis compositions, and particular sequences of the gp19compositions may impart its immunogenicity; for example, a region of thegp19 composition may comprise an epitope. In particular embodiments, oneor more epitopes on a gp19 composition are located in the C-terminus orin the N-terminus of a gp19 polypeptide. In specific aspects, theC-terminus comprises the last 60 amino acids of SEQ ID NO:17 or SEQ IDNO:19, for example. In additional aspects of the invention, theC-terminus comprises the last 60 amino acids of SEQ ID NO:17 or SEQ IDNO:19, and in particular aspects the C-terminus comprises the last 55,the last 50, the last 45, the last 40, the last 35, the last 30, thelast 25, the last 20, the last 15, the last 10, or the last 5 aminoacids of SEQ ID NO:17 or SEQ ID NO:19. In additional aspects of theinvention, the C-terminus comprises no more than the last 60 amino acidsof SEQ ID NO:17 or SEQ ID NO:19, and in particular aspects theC-terminus comprises no more than the last 55, the last 50, the last 45,the last 40, the last 35, the last 30, the last 25, the last 20, thelast 15, the last 10, or the last 5 amino acids of SEQ ID NO:17 or SEQID NO:19. In other specific aspects, the N-terminus comprises the first74 amino acids of SEQ ID NO:17 or SEQ ID NO:19. In further aspects ofthe invention, the N-terminus comprises the first 74 amino acids of SEQID NO:17 or SEQ ID NO:19, and in particular aspects the N-terminuscomprises the first 70, the first 65, the first 60, the first 55, thefirst 50, the first 45, the first 40, the first 35, the first 30, thefirst 25, the first 20, the first 15, the first 10, or the first 5 aminoacids of SEQ ID NO:17 or SEQ ID NO:19. In further aspects of theinvention, the N-terminus comprises no more than the first 74 aminoacids of SEQ ID NO:17 or SEQ ID NO:19, and in particular aspects theN-terminus comprises no more than the first 70, the first 65, the first60, the first 55, the first 50, the first 45, the first 40, the first35, the first 30, the first 25, the first 20, the first 15, the first10, or the first 5 amino acids of SEQ ID NO:17 or SEQ ID NO:19.

In some aspects of the invention, multiple different E. canis strainscomprise immunogenic gp19 compositions, and there is significantsequence identity among the strains in regions of the gp19 compositionsthat comprise the epitope (such as greater than about 80%, 85%, 90%,95%, or 98%, for example). However, in some embodiments, there may besignificant sequence identity among the strains in regions of the gp19compositions that do not comprise the epitope. In particular aspects ofthe invention, there is a gp19 composition that is immunogenic for morethan one strain of E. canis, including, for example, North Carolina(Jake), Oklahoma, North Carolina (Demon), North Carolina (DJ), NorthCarolina (Fuzzy), Louisiana, Florida, and in particular aspects theepitope of the other strains is SEQ ID NO:13, although other epitopesmay also be identified. In embodiments wherein an alternative gp19 E.canis epitope to SEQ ID NO:13 is identified, there may be provided animmunogenic composition comprising a mixture of gp19 E. canis epitopes,such as a mixture including SEQ ID NO:13, for example.

In certain embodiments of the invention, immunogenic compositions of E.canis comprise one or more carbohydrate moieties. In particular aspects,the carbohydrate moieties facilitate the immunogenic nature of thecomposition. In specific embodiments, the carbohydrate moiety isrequired for immunogenicity, whereas in alternative embodiments thecarbohydrate moiety enhances immunogenicity. The carbohydrate moiety maybe of any kind, so long as it is suitable to allow or enhanceimmunogenicity. The identity of a carbohydrate moiety may be determinedby any suitable means in the art, although in particular aspects anenzyme that cleaves particular carbohydrates from polypeptides orpeptides, followed by analysis of the cleaved carbohydrate, for examplewith mass spectroscopy, may be utilized. In other means, thecarbohydrate is removed and assayed with a variety of lectins, which areknown to bind specific sugars. In specific embodiments, the carbohydratecomprises glucose, galactose and/or xylose. In specific embodiments ofthe invention, one or more carbohydrate moieties on the glycoprotein areidentified by suitable method(s) in the art, for example gaschromatography/mass spectrometry.

In an embodiment of the invention, there is an immunogenic gp19 E. canisglycoprotein. In an additional embodiment of the invention, there is anE. canis composition comprising SEQ ID NO:13. In specific aspects of theinvention, the composition further comprises a pharmaceuticallyacceptable excipient. The composition may be further defined ascomprising one or more carbohydrate moieties, as comprising part or allof an epitope, and/or as a vaccine, such as a subunit vaccine.

In another embodiment of the invention, there is an E. canis compositioncomprising a polypeptide encoded by at least part of the polynucleotideof SEQ ID NO:16 or SEQ ID NO:18 and/or an E. canis compositioncomprising a polypeptide of SEQ ID NO:17 or SEQ ID NO:19. In oneembodiment of the invention, there is an isolated composition comprisingan Ehrlichia gp19 glycoprotein, comprising: (a) a sequence selected fromthe group consisting of SEQ ID NO:13, SEQ ID NO:17, or SEQ ID NO:19; or(b) a sequence that is at least about 70% identical to one or moresequences in (a). The composition may be further defined as a sequencethat is at least about 75%, about 80%, about 85%, about 90%, or about95% identical to one or more sequences in (a). The composition may alsobe further defined as being comprised in a pharmaceutically acceptableexcipient, as comprising one or more carbohydrate moieties, and/or asbeing comprised in a pharmaceutical composition suitable as a vaccine.

In a specific embodiment, there is an isolated polynucleotide thatencodes SEQ ID NO:17, an isolated polynucleotide that encodes SEQ IDNO:19, an isolated polynucleotide that encodes SEQ ID NO:13, or amixture thereof.

In particular embodiments, there is an isolated polynucleotide,comprising: a) a polynucleotide that encodes SEQ ID NO:17; or b) apolynucleotide that is at least about 90% identical to thepolynucleotide of a) and that encodes an immunoreactive E. canis gp19polypeptide. In a specific embodiment, the polynucleotide is furtherdefined as SEQ ID NO:16.

In particular embodiments, there is an isolated polynucleotide,comprising: a) a polynucleotide that encodes SEQ ID NO:19; or b) apolynucleotide that is at least about 90% identical to thepolynucleotide of a) and that encodes an immunoreactive E. canis gp19polypeptide. In a specific embodiment, the polynucleotide is furtherdefined as SEQ ID NO:18.

In a further embodiment of the invention, there is an isolatedpolynucleotide, comprising: a) a polynucleotide that encodes SEQ IDNO:17; or b) a polynucleotide that is at least about 90% identical tothe polynucleotide of a) and that encodes an immunoreactive E. canisgp19 polypeptide. In a specific embodiment, the polynucleotide isfurther defined as SEQ ID NO:16. In additional aspects of the invention,there is an isolated polynucleotide, comprising: a) a polynucleotidethat encodes SEQ ID NO:19; or b) a polynucleotide that is at least about90% identical to the polynucleotide of a) and that encodes animmunoreactive E. canis gp19 polypeptide. In a specific embodiment thepolynucleotide is further defined as SEQ ID NO:18.

In an additional embodiment of the invention, there is an isolatedpolypeptide, comprising: a) SEQ ID NO:17 and/or SEQ ID NO:19; or b) agp19 polypeptide that is at least about 70% identical to SEQ ID NO:17and/or SEQ ID NO:19 and that comprises immunogenic activity. In aspecific embodiment, the polypeptide is comprised in a pharmaceuticallyacceptable excipient, and/or it may be further defined as beingcomprised in a pharmaceutical composition suitable as a vaccine.

In certain aspects of the invention, there are polynucleotides that areamplifiable by one or more of the exemplary primers of SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:14, or SEQ ID NO:15.

In specific aspects of the invention, there is a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:13, and in specificembodiments the polynucleotide comprises SEQ ID NO:20. In other aspectsof the invention, there is a polynucleotide that encodes a polypeptidecomprising SEQ ID NO:17 and/or SEQ ID NO:19.

In another aspect of the invention, there are isolated antibodies thatbind one or more polypeptides of the invention. Antibodies may bemonoclonal, polyclonal, or antibody fragments, for example. Inparticular embodiments, the antibody binds selectively to an epitope ofgp19, for example one that comprises SEQ ID NO:13. In specificembodiments, the antibody may be referred to as immunologically reactingwith one or more polypeptides of the invention.

In further aspects, there is a peptide or polypeptide that comprises SEQID NO:13 or has a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 substitutions relativeto SEQ ID NO:13, and in specific aspects the substitutions areconservative substitutions.

In an additional embodiment of the invention, there is a method ofproviding resistance to E. canis infection, comprising the step ofdelivering a therapeutically effective amount of a composition of theinvention, such as a gp19 antibody, polypeptide, and/or polynucleotide,to the individual.

In another embodiment, there is a method of inducing an immune responsein an individual, comprising the step of delivering to the individual atherapeutically effective amount of a gp19 polypeptide of the invention.In an additional embodiment of the present invention, there is a methodof inhibiting or preventing E. canis infection in a subject comprisingthe steps of: identifying a subject prior to exposure or suspected ofbeing exposed to or infected with E. canis; and administering apolypeptide, antibody, and/or polynucleotide of the invention in anamount effective to inhibit E. canis infection.

In some aspects of the invention the composition may be encoded by apolynucleotide comprising: (a) a polynucleotide selected from the groupconsisting of SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:20; or (b) apolynucleotide that is at least about 70% identical to a polynucleotideof (a) and encodes an immunoreactive E. canis gp19 polypeptide; or (c) apolynucleotide that hybridizes to one or more polynucleotides of (a) or(b) under stringent conditions. In specific embodiments of theinvention, the polynucleotide of (c) is at least about 70% identical, atleast about 75% identical, at least about 80% identical, at least about85% identical, at least about 90% identical, or at least about 95%identical to a polynucleotide of (a) or (b) and encodes animmunoreactive E. canis gp19 polypeptide.

Polynucleotides of the invention may be comprised in a vector, such as aviral vector or a non-viral vector, wherein the viral vector may be anadenoviral vector, a retroviral vector, a lentiviral vector, anadeno-associated vector, a herpes virus vector, or a vaccinia virusvector and wherein the non-viral vector may be a plasmid. In furtheraspects of the invention, the vector comprise a promoter operably linkedto the polynucleotide wherein the promoter is operable in a prokaryote,a eukaryote, or both. The polynucleotide of the invention may becomprised in a liposome and/or comprised in a pharmaceuticallyacceptable excipient.

In certain aspects of the invention, there is an isolated antibody thatreacts immunologically to a polypeptide of the invention, and theantibody may be a monoclonal antibody, may be comprised in polyclonalantisera, or may be an antibody fragment, for example.

In other embodiments of the invention, there is a method of inducing animmune response in an individual, comprising the step of delivering tothe individual a therapeutically effective amount of a composition ofthe invention, such as a polypeptide, antibody and/or polynucleotide.

In additional embodiments of the invention, there is a method ofinhibiting E. canis infection in a subject comprising the steps of:identifying a subject prior to exposure or suspected of being exposed toor infected with E. canis; and administering the polypeptide of theinvention in an amount effective to inhibit E. canis infection. Infurther embodiments of the invention, there is a method of identifyingan E. canis infection in an individual, comprising the step of assayinga sample from the individual for an antibody, polypeptide, and/orpolynucleotide of the invention.

In one embodiment of the invention, there is a pharmaceuticalcomposition, comprising one or more of the following: (a) an isolatedpolypeptide comprising SEQ ID NO:17 or SEQ ID NO:19; (b) an isolatedpolypeptide that is at least 70% identical to a polypeptide of (a); (c)an isolated polypeptide comprising SEQ ID NO:13; or (d) an isolatedpolypeptide that is at least 70% identical to SEQ ID NO:13, wherein saidpolypeptide is dispersed in a pharmaceutically acceptable diluent. Inspecific embodiments, (b) is further defined as a polypeptide that is atleast 75% identical to a polypeptide of (a); as a polypeptide that is atleast 80% identical to a polypeptide of (a); as a polypeptide that is atleast 85% identical to a polypeptide of (a); as a polypeptide that is atleast 90% identical to a polypeptide of (a); or as a polypeptide that isat least 95% identical to a polypeptide of (a). The pharmaceuticalcomposition may further defined as a vaccine composition.

In specific embodiments, a polypeptide of a pharmaceutical compositionis further defined as comprising one or more carbohydrate moieties. Incertain aspects, the polypeptide of comprises SEQ ID NO:17 or thepolypeptide comprises SEQ ID NO:19.

In specific aspects of the invention, a polypeptide is further definedas being from 24 to 30 amino acids in length, from 24 to 35 amino acidsin length, from 24 to 40 amino acids in length, from 24 to 45 aminoacids in length, from 24 to 50 amino acids in length, from 24 to 55amino acids in length, from 24 to 60 amino acids in length, from 24 to65 amino acids in length, from 24 to 70 amino acids in length, from 24to 75 amino acids in length, from 24 to 80 amino acids in length, from24 to 85 amino acids in length, from 24 to 90 amino acids in length,from 24 to 95 amino acids in length, or from 24 to 100 amino acids inlength, for example.

Variants of polypeptides comprising SEQ ID NO:13 may be defined as beingat least 80% identical to SEQ ID NO:13; as being at least 85% identicalto SEQ ID NO:13; as being at least 90% identical to SEQ ID NO:13; or asbeing at least 95% identical to SEQ ID NO:13.

In additional embodiments of the invention, there is a pharmaceuticalcomposition comprising an isolated polypeptide encoded by an isolatednucleic acid molecule, said nucleic acid molecule comprising: (a) apolynucleotide comprising SEQ ID NO:16 or SEQ ID NO:18; or (b) apolynucleotide that is capable of hybridizing under stringent conditionsto the polynucleotide of (a); wherein the polypeptide has at least 70%identity to SEQ ID NO:17 or SEQ ID NO:19 and wherein the polypeptide isdispersed in a pharmaceutically acceptable diluent. The polypeptide maybe at least 75% identical to SEQ ID NO:17 or SEQ ID NO:19; at least 80%identical to SEQ ID NO:17 or SEQ ID NO:19; at least 85% identical to SEQID NO:17 or SEQ ID NO:19; at least 90% identical to SEQ ID NO:17 or SEQID NO:19; or at least 95% identical to SEQ ID NO:17 or SEQ ID NO:19.

The invention in certain aspects concerns a composition, comprising (a)an isolated polypeptide or peptide comprising more than 15, such as morethan 20, such as more then 23, but no more than 130 contiguous aminoacids of SEQ ID NO:17 or SEQ ID NO:19; or (b) a polypeptide or peptidethat is about 70%, about 75%, about 80%, about 85%, about 90%, or about95% identical to a sequence that is no more than 130 contiguous aminoacids of SEQ ID NO:17 or SEQ ID NO:19.

In additional aspects, there is a polypeptide further defined as beingencoded by a polynucleotide that is no more than 75%, 80%, 85%, 90%,95%, 97%, or 99% identical to SEQ ID NO:16 or SEQ ID NO:18.

In an additional embodiment, there is a composition comprising: (a) apeptide having SEQ ID NO:13; or (b) a variant of the peptide of (a),wherein the variant is at least 75% identical to SEQ ID NO:13, whereinthe composition is capable of eliciting an immune reaction in anindividual. In a specific embodiment, there is a peptide is from 24 to50 amino acids in length. In a specific embodiment, there is a variantis further defined as being at least 80%, at least 85%, at least 90%, orat least 95% identical to SEQ ID NO:13.

A composition of the invention may be defined as having activity thatprovides immunity against Ehrlichia canis for an individual. Acomposition of the invention may be defined as having activity thatinduces an immune reaction against Ehrlichia canis for an individual.Compositions of the invention include any polypeptide, peptide,polynucleotide, and/or antibody provided herein.

In another embodiment of the invention, there is an isolated nucleicacid molecule, comprising: (a) a polynucleotide comprising SEQ ID NO:16or SEQ ID NO:18; or (b) a polynucleotide that is capable of hybridizingunder stringent conditions to the polynucleotide of (a) and that encodesa polypeptide having at least 70% identity to SEQ ID NO:17 or SEQ IDNO:19, wherein said nucleic acid molecule is operably linked to aheterologous promoter, such as a promoter that is active in a eukaryoticcell or that is active in a prokaryotic cell. In a specific embodiment,the nucleic acid molecule is further defined as the polynucleotidecomprising SEQ ID NO:16 or as the polynucleotide comprising SEQ IDNO:18. The polynucleotide of (b) may be further defined as apolynucleotide that encodes a polypeptide that is at least 75% identicalto SEQ ID NO:17 or SEQ ID NO:19; that is at least 80% identical to SEQID NO:17 or SEQ ID NO:19; that is at least 85% identical to SEQ ID NO:17or SEQ ID NO:19; that is at least 90% identical to SEQ ID NO:17 or SEQID NO:19; or that is at least 95% identical to SEQ ID NO:17 or SEQ IDNO:19.

In an additional embodiment of the invention, there is an isolated DNA,comprising: (a) sequence that is no less than 75% but no more than 98%identical to SEQ ID NO:16 or SEQ ID NO:18; or (b) sequence that iscomplementary to the sequence in (a). In specific embodiments, (a) isfurther defined as a sequence that is no less than 80% but no more than98% identical to SEQ ID NO:16 or SEQ ID NO:18; as a sequence that is noless than 85% but no more than 98% identical to SEQ ID NO:16 or SEQ IDNO:18; as a sequence that is no less than 90% but no more than 98%identical to SEQ ID NO:16 or SEQ ID NO:18; as a sequence that is no lessthan 95% but no more than 98% identical to SEQ ID NO:16 or SEQ ID NO:18;as a sequence that is no less than 80% but no more than 95% identical toSEQ ID NO:16 or SEQ ID NO:18; as a sequence that is no less than 80% butno more than 90% identical to SEQ ID NO:16 or SEQ ID NO:18; or as asequence that is no less than 80% but no more than 85% identical to SEQID NO:16 or SEQ ID NO:18.

Nucleic acid molecules may be further defined as being comprised in avector, such as a viral vector or a non-viral vector, wherein the viralvector may comprise an adenoviral vector, a retroviral vector, or anadeno-associated viral vector. The nucleic acid molecule may becomprised in a liposome.

In specific embodiments, there is an isolated antibody thatimmunologically reacts with one or more of the amino acid sequencesselected from the group consisting of SEQ ID NO:13, SEQ ID NO:17, andSEQ ID NO:19. In further specific embodiments, the antibody is amonoclonal antibody, is comprised in polyclonal antisera, or is anantibody fragment.

In an additional embodiment, there is a method of producing apolypeptide, comprising: providing a host cell comprising apolynucleotide of the invention and culturing the cell under conditionssuitable for the host cell to express the polynucleotide to produce theencoded polypeptide. The method may further comprise isolating thepolypeptide.

In another embodiment, there is a method of producing a polynucleotide,comprising: hybridizing SEQ ID NO:16 or SEQ ID NO:18 to genomic DNAunder stringent conditions; and isolating the polynucleotide detectedwith SEQ ID NO:16 or SEQ ID NO:18. In a specific embodiment, there is anisolated DNA prepared according to the method.

In an additional embodiment of the invention, there is a method ofinducing an immune response in an individual, comprising the step ofdelivering to the individual a therapeutically effective amount of acomposition of the invention.

In a further embodiment of the invention, there is a method ofinhibiting E. canis infection in a subject, comprising the step ofadministering to the subject prior to exposure or suspected of beingexposed to or infected with E. canis, an effective amount of acomposition of the invention.

In an additional embodiment of the invention, there is a method ofidentifying an E. canis infection in an individual, comprising the stepof assaying a sample from the individual for one or both of thefollowing: (a) a polypeptide of SEQ ID NO:17, SEQ ID NO:19, or both; or(b) an antibody that immunologically reacts with an amino acid sequenceselected from the group consisting of SEQ ID NO:13, SEQ ID NO:17, andSEQ ID NO:19. In a specific embodiment of this method, the polypeptideof (a) is SEQ ID NO:17. In a specific embodiment of this method, thepolypeptide of (a) is SEQ ID NO:19. In a specific embodiment of thismethod, the polypeptide of (a) is a mixture of SEQ ID NO:17 and SEQ IDNO:19. In specific embodiments, the antibody of (b) immunologicallyreacts with an amino acid sequence of SEQ ID NO:13, SEQ ID NO:17, or SEQID NO:19. In specific aspects, assaying a sample for an antibody isfurther defined as assaying for an antibody by ELISA, such as byallowing assaying for one or more E. canis antibodies other then theantibody of (b). The other E. canis antibodies are selected from thegroup consisting of antibodies for gp36, gp19, gp28/30, and gp200.

In an embodiment of the invention, there is a kit, comprising one ormore of the following compositions: (a) an isolated polypeptidecomprising SEQ ID NO:17 or SEQ ID NO:19; (b) an isolated polypeptidethat is at least 70% identical to a polypeptide of (a); (c) an isolatedpolypeptide comprising SEQ ID NO:13; (d) an isolated polypeptide that isat least 70% identical to SEQ ID NO:13; (e) a polynucleotide comprisingSEQ ID NO:16 or SEQ ID NO:18; (f) a polynucleotide that is capable ofhybridizing under stringent conditions to the polynucleotide of (a) andthat encodes a polypeptide having at least 70% identity to SEQ ID NO:17or SEQ ID NO:19; or (g) an isolated antibody that immunologically reactswith one or more of the amino acid sequences selected from the groupconsisting of SEQ ID NO:13, SEQ ID NO:17, and SEQ ID NO:19. In aspecific embodiment, the kit is further defined as comprising two ormore of the compositions.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features that are believed to be characteristic of theinvention, both as to its organization and method of operation, togetherwith further objects and advantages will be better understood from thefollowing description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.

FIG. 1 provides a schematic of E. canis gp19 chromosomal location andadjacent genes (size in bp) and intergenic regions (size in bp). E.chaffeensis vlpt had the same adjacent genes.

FIG. 2 relates to gp19 thiofusion protein. (Panel A) Molecular mass ofE. canis gp19 pBAD thiofusion protein (˜35 kDa) (lane 1) after sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); M-BioRadPrecision molecular weight marker. (Panel B) Corresponding Westernimmunoblot of recombinant gp19 thiofusion protein (lane 1) andthioredoxin control protein (13-kDa) (lane 2) reacted with anti-E. canisdog serum (#2995).

FIG. 3 shows carbohydrate detection of with E. canis gp19 (aminoterminal fragment) (lane 2) and E. canis Dsb protein (lane 1; negativecontrol). M=BioRad Precision Protein Standards; CCM=CandyCaneglycoprotein molecular weight standards containing a mixture ofglycosylated and non-glycosylated proteins (Glycosylated proteins, 42-and 18-kDa; non-glycosylated proteins, 29- and 14-kDa).

FIG. 4 shows westerns related to gp19. (Panel A) Western immunoblot ofE. canis whole cell lysates probed with anti-E. canis gp19 serum(lane 1) and anti-E. canis dog serum (lane 2): Infected DH82 celllysates probed with anti-E. canis dog serum (lane 3). (Panel B) E.chaffeensis whole cell lysates probed with anti-E. canis gp19 (lane 1)and anti-E. chaffeensis dog serum (lane 2).

FIG. 5 provides (Top) schematic of E. canis recombinant gp19 fragmentsincluding the epitope-comprising region N1-C. (Panel A) SDS-PAGE of E.canis recombinant gp19 fragments (N1, lane 1; N2, lane 2; N-terminal,lane 3; and C-terminal, lane 4; and thioredoxin control) andcorresponding Western immunoblot probed with anti-E. canis dog serum(Panel B).

FIG. 6 shows immunoreactivity of recombinant gp19 (N1-C epitope;glycosylated) with canine anti-E. canis serum compared to the syntheticpeptide (aglycosylated) by ELISA (top). Immunoreactivity of E. canisgp19 (N1-C epitope) with anti-E. canis dog serum after treatment withperiodate as determined by ELISA (bottom).

FIG. 7 demonstrates an exemplary immunogold-labeled electronphotomicrograph of E. canis gp19 localization in a morula containingboth reticulate and dense-cored Ehrlichiae.

FIG. 8 shows an exemplary confocal immunofluorescent photomicrograph ofE. canis gp19 expression. E. canis infected cells were dually stainedwith anti-E. canis gp19 (red; left) and with anti-ehrlichial Dsb (green;center) and merged images (right).

FIG. 9 shows demonstration of the kinetics of IgG antibody responses toE. canis in three experimentally infected dogs (A=Dog 33; B=Dog 34; andC=Dog 44) to five recombinant proteins gp36 (⋄), gp19 (□), p28 (Δ),gp200N (x), gp200C (∘) and a thioredoxin control (+) on days 0, 7, 14,21, 28, 35, and 42 post inoculation as determined by Western immunoblot(left) and corresponding ELISA (right).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

In keeping with long-standing patent law convention, the words “a” and“an” when used in the present specification in concert with the wordcomprising, including the claims, denote “one or more.” Some embodimentsof the invention may consist of or consist essentially of one or moreelements, method steps, and/or methods of the invention. It iscontemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein.

The term “carbohydrate” as used herein refers to a composition comprisedof carbon, hydrogen, and oxygen, particularly in the ratio of 2H:1C:1O.The term includes sugars, starches, and celluloses, for example.

The term “epitope” as used herein refers to a site of a composition towhich a specific antibody binds.

The term “glycan,” which may also be referred to as a “polysaccharide,”as used herein refers to a carbohydrate that can be decomposed byhydrolysis into two or more monosaccharides. In other words, it may bereferred to as a chain of simple sugars (aldehyde or ketone derivativesof a polyhydric alcohol).

The term “identity” as known in the art, refers to a relationshipbetween the sequences of two or more polypeptide molecules or two ormore nucleic acid molecules, as determined by comparing the sequences.In the art, “identity” also means the degree of sequence relatednessbetween nucleic acid molecules or between polypeptides, as the case maybe, as determined by the number of matches between strings of two ormore nucleotide residues or two or more amino acid residues. “Identity”measures the percent of identical matches between the smaller of two ormore sequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”).

The term “immunogenic” as used herein refers to a composition that isable to provoke an immune response against it.

The term “immune response” as used herein refers to the reaction of theimmune system to the presence of an antigen by making antibodies to theantigen. In further specific embodiments, immunity to the antigen may bedeveloped on a cellular level, by the body as a whole, hypersensitivityto the antigen may be developed, and/or tolerance may be developed, suchas from subsequent challenge. In specific embodiments, an immuneresponse entails lymphocytes identifying an antigenic molecule asforeign and inducing the formation of antibodies and lymphocytes capableof reacting with it and rendering it less harmful.

The term “immunoreactive” as used herein refers to a composition beingreactive with antibodies from the sera of an individual. In specificembodiments, a composition is immunoreactive if an antibody recognizesit, such as by binding to it and/or immunologically reacting with it.

The term “mucin” as used herein refers to one or more highlyglycosylated glycoproteins with N-acetylgalactosamine (GalNAc.)

The term “ortholog” as used herein refers to a polynucleotide from onespecies that corresponds to a polynucleotide in another species; the twopolynucleotides are related through a common ancestral species (ahomologous polynucleotide). However, the polynucleotide from one specieshas evolved to become different from the polynucleotide of the otherspecies.

The term “similarity” is a related concept, but in contrast to“identity”, refers to a sequence relationship that includes bothidentical matches and conservative substitution matches. If twopolypeptide sequences have, for example, 10/20 identical amino acids,and the remainder are all non-conservative substitutions, then thepercent identity and similarity would both be 50%. If, in the sameexample, there are 5 more positions where there are conservativesubstitutions, then the percent identity remains 50%, but the percentsimilarity would be 75% ( 15/20). Therefore, in cases where there areconservative substitutions, the degree of similarity between twopolypeptides will be higher than the percent identity between those twopolypeptides.

The term “subunit vaccine” as used herein refers to a vaccine wherein apolypeptide or fragment thereof is employed, as opposed to an entireorganism.

The term “vaccine” as used herein refers to a composition that providesimmunity to an individual upon challenge.

The term “virulence factor” as used herein refers to one or more geneproducts that enable a microorganism to establish itself on or within aparticular host species and enhance its pathogenicity. Exemplaryvirulence factors include, for example, cell surface proteins thatmediate bacterial attachment, cell surface carbohydrates and proteinsthat protect a bacterium, bacterial toxins, and hydrolytic enzymes thatmay contribute to the pathogenicity of the bacterium.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and so forth which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.,Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL,Second Edition (1989), OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait Ed., 1984),ANIMAL CELL CULTURE (R. I. Freshney, Ed., 1987), the series METHODS INENZYMOLOGY (Academic Press, Inc.); GENE TRANSFER VECTORS FOR MAMMALIANCELLS (J. M. Miller and M. P. Calos eds. 1987), HANDBOOK OF EXPERIMENTALIMMUNOLOGY, (D. M. Weir and C. C. Blackwell, Eds.), CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore,J. G. Siedman, J. A. Smith, and K. Struhl, eds., 1987), CURRENTPROTOCOLS IN IMMUNOLOGY (J. E. coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach and W. Strober, eds., 1991); ANNUAL REVIEW OFIMMUNOLOGY; as well as monographs in journals such as ADVANCES INIMMUNOLOGY. All patents, patent applications, and publications mentionedherein, both supra and infra, are hereby incorporated herein byreference.

II. Embodiments of the Present Invention

The present invention concerns compositions and methods related toEhrlichia spp. proteins and the polynucleotides that encode them. Inparticular aspects of the invention, there are differentially-expressedand secreted major immunoreactive protein orthologs of E. canis and E.chaffeensis that elicit early antibody responses to epitopes onglycosylated tandem repeats. Specifically, the present inventionconcerns one or more glycoproteins from Ehrlichia spp., in specificembodiments. In further embodiments, the present invention relates to aglycoprotein from Ehrlichia spp. that is a gp19 protein. In additionalembodiments, the gp19 protein is from E. canis.

Ehrlichia canis has a small subset of major immunoreactive proteins thatincludes a 19-kDa protein that elicits an early ehrlichial specificantibody response in infected dogs. The present invention concerns theidentification and molecular characterization of this highly conserved19-kDa major immunoreactive glycoprotein (gp19) ortholog of the E.chaffeensis variable-length PCR target (VLPT) protein. The E. canis gp19has substantial carboxyl-terminal amino acid homology (59%) with E.chaffeensis VLPT and the same chromosomal location; however, the E.chaffeensis vlpt gene (594-bp) has tandem repeats that are not presentin the E. canis gp19 (414-bp). Consistent with other ehrlichialglycoproteins, the gp19 exhibited a larger than predicted mass (˜3 kDa),O-linked glycosylation sites were predicted in an amino-terminalserine/threonine/glutamate (STE)-rich patch (24 amino acids),carbohydrate was detected on the recombinant gp19, and neutral sugarsglucose and xylose were detected on the recombinant amino-terminalregion. The E. canis gp19 composition comprises five predominant aminoacids, cysteine, glutamate, tyrosine, serine and threonine, concentratedin the STE-rich patch and within a carboxyl-terminal tail predominatedby cysteine and tyrosine (55%). The amino-terminal STE-rich patchcomprised a major species-specific antibody epitope strongly recognizedby sera from an E. canis-infected dog. An exemplary recombinantglycopeptide epitope was substantially more reactive with antibody thanan exemplary synthetic (nonglycosylated) peptide, and periodatetreatment of the recombinant glycopeptide epitope reduced itsimmunoreactivity, indicating that carbohydrate is useful as part of animmunodeterminant. The gp19 was present on reticulate and dense coredcells and it was found extracellularly in the fibrillar matrix andassociated with the morula membrane.

Some embodiments of the present invention are directed toward a methodof inhibiting E. canis infection in a subject comprising the steps ofidentifying a subject prior to exposure or suspected of being exposed toor infected with E. canis and administering a composition comprising a19-kDa antigen of E. canis in an amount effective to inhibit E. canisinfection. The inhibition may occur through any means such as e.g., thestimulation of the subject's humoral or cellular immune responses, or byother means such as inhibiting the normal function of the 19-kDaantigen, or even competing with the antigen for interaction with someagent in the subject's body, or a combination thereof, for example.

The present invention is also directed toward a method of targetedtherapy to an individual, comprising the step of administering acompound to an individual, wherein the compound has a targeting moietyand a therapeutic moiety, and wherein the targeting moiety is specificfor gp19 protein. In certain aspects, the targeting moiety is anantibody specific for gp19 or ligand or ligand binding domain that bindsgp19. Likewise, the therapeutic moiety may comprise a radioisotope, atoxin, a chemotherapeutic agent, an immune stimulant, a cytotoxic agent,or an antibiotic, for example.

Other embodiments of the present invention concern diagnosis ofehrlichial infection in a mammal by assaying a sample from the mammal,such as blood or serum, for example, for antibodies to a gp19composition (for E. canis).

III. E. canis gp19 Amino Acid Compositions

The present invention regards a polypeptide or peptide comprising E.canis gp19. For the sake of brevity, the following section will refer toany E. canis gp19 amino acid compositions of the present invention,including polypeptides and peptides.

In particular embodiments, a polypeptide may be a recombinantpolypeptide or it may be isolated and/or purified from nature, forexample. In particular aspects, the amino acid sequence is encoded by anucleic acid sequence. The polypeptide is useful as an antigen, inspecific embodiments. In other particular embodiments, a peptide may begenerated synthetically or encoded by an oligonucleotide, for example.The peptide is useful as an antigen, in specific embodiments.

The present invention is also directed towards a method of producing therecombinant polypeptide, comprising the steps of obtaining a vector thatcomprises an expression construct comprising a sequence encoding theamino acid sequence operatively linked to a promoter; transfecting thevector into a cell; and culturing the cell under conditions effectivefor expression of the expression construct. The amino acid sequence maybe generated synthetically, in alternative embodiments.

By a “substantially pure protein” is meant a protein that has beenseparated from at least some of those components that naturallyaccompany it. A substantially pure immunoreactive composition may beobtained, for example, by extraction from a natural source; byexpression of a recombinant nucleic acid encoding an immunoreactivecomposition; or by chemically synthesizing the protein, for example.Accordingly, substantially pure proteins include proteins synthesized inE. coli, other prokaryotes, or any other organism in which they do notnaturally occur.

Thus, in certain embodiments, the present invention concerns novelcompositions comprising at least one proteinaceous molecule. As usedherein, a “proteinaceous molecule,” “proteinaceous composition,”“proteinaceous compound,” “proteinaceous chain” or “proteinaceousmaterial” generally refers, but is not limited to, a protein of greaterthan about 130 amino acids or the full length endogenous sequencetranslated from a gene; a polypeptide of greater than about 100 aminoacids; and/or a peptide of from about 3 to about 100 amino acids. Allthe “proteinaceous” terms described above may be used interchangeablyherein.

In certain embodiments the size of the at least one proteinaceousmolecule may comprise, but is not limited to, about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30, about 31, about32, about 33, about 34, about 35, about 36, about 37, about 38, about39, about 40, about 41, about 42, about 43, about 44, about 45, about46, about 47, about 48, about 49, about 50, about 51, about 52, about53, about 54, about 55, about 56, about 57, about 58, about 59, about60, about 61, about 62, about 63, about 64, about 65, about 66, about67, about 68, about 69, about 70, about 71, about 72, about 73, about74, about 75, about 76, about 77, about 78, about 79, about 80, about81, about 82, about 83, about 84, about 85, about 86, about 87, about88, about 89, about 90, about 91, about 92, about 93, about 94, about95, about 96, about 97, about 98, about 99, about 100, about 110, about120, about 130, or greater amino acid residues, and any range derivabletherein.

As used herein, an “amino acid molecule” refers to any polypeptide,polypeptide derivitive, or polypeptide mimetic as would be known to oneof ordinary skill in the art. In certain embodiments, the residues ofthe proteinaceous molecule are sequential, without any non-amino acidmolecule interrupting the sequence of amino acid molecule residues. Inother embodiments, the sequence may comprise one or more non-aminomolecule moieties. In particular embodiments, the sequence of residuesof the proteinaceous molecule may be interrupted by one or morenon-amino molecule moieties.

Accordingly, the term “proteinaceous composition” encompasses aminomolecule sequences comprising at least one of the 20 common amino acidsin naturally synthesized proteins, or at least one modified or unusualamino acid, including but not limited to those shown on Table 1 below.

TABLE 1 Modified and Unusual Amino Acids Abbr. Amino Acid Aad2-Aminoadipic acid Baad 3-Aminoadipic acid Bala β-alanine,β-Amino-propionic acid Abu 2-Aminobutyric acid 4Abu 4-Aminobutyric acid,piperidinic acid Acp 6-Aminocaproic acid Ahe 2-Aminoheptanoic acid Aib2-Aminoisobutyric acid Baib 3-Aminoisobutyric acid Apm 2-Aminopimelicacid Dbu 2,4-Diaminobutyric acid Des Desmosine Dpm 2,2′-Diaminopimelicacid Dpr 2,3-Diaminopropionic acid EtGly N-Ethylglycine EtAsnN-Ethylasparagine Hyl Hydroxylysine AHyl allo-Hydroxylysine 3Hyp3-Hydroxyproline 4Hyp 4-Hydroxyproline Ide Isodesmosine AIleallo-Isoleucine MeGly N-Methylglycine, sarcosine MeIleN-Methylisoleucine MeLys 6-N-Methyllysine MeVal N-Methylvaline NvaNorvaline Nle Norleucine Orn Ornithine

In certain embodiments the proteinaceous composition comprises at leastone protein, polypeptide or peptide. In further embodiments, theproteinaceous composition comprises a biocompatible protein, polypeptideor peptide. As used herein, the term “biocompatible” refers to asubstance that produces no significant untoward effects when applied to,or administered to, a given organism according to the methods andamounts described herein. Such untoward or undesirable effects are thosesuch as significant toxicity or adverse immunological reactions.

Proteinaceous compositions may be made by any technique known to thoseof skill in the art, including the expression of proteins, polypeptidesor peptides through standard molecular biological techniques, theisolation of proteinaceous compounds from natural sources, or thechemical synthesis of proteinaceous materials, for example. Thenucleotide and protein, polypeptide and peptide sequences for variousgenes have been previously disclosed, and may be found at computerizeddatabases known to those of ordinary skill in the art. Two suchdatabases are the National Center for Biotechnology Information'sGenBank® and GenPept databases, for example. The coding regions forthese known genes may be amplified and/or expressed using the techniquesdisclosed herein or as would be know to those of ordinary skill in theart. Alternatively, various commercial preparations of proteins,polypeptides and peptides are known to those of skill in the art.

In certain embodiments a proteinaceous compound may be purified.Generally, “purified” will refer to a specific or protein, polypeptide,or peptide composition that has been subjected to fractionation toremove various other proteins, polypeptides, or peptides, and whichcomposition substantially retains its activity, as may be assessed, forexample, by the protein assays, as would be known to one of ordinaryskill in the art for the specific or desired protein, polypeptide orpeptide. Exemplary activities that may be assessed for retention in thepurified proteinaceous composition are iron-binding activity andimmunoreactivity.

In specific embodiments of the present invention, a polypeptide islabeled, and any detectable label is suitable in the invention. Thelabel may be attached to the polypeptide at the N-terminus, at theC-terminus, or in a side chain of an amino acid residue, for example.One or more labels may be employed. Exemplary labels includedradioactive labels, fluorescent labels, colorimetric labels, and soforth. In specific embodiments, the label is covalently attached to thepolypeptide.

IV. E. canis gp19 Nucleic Acid Compositions

Certain embodiments of the present invention concern an E. canis gp19nucleic acid. For the sake of brevity, the following section will referto any E. canis gp19 nucleic acid compositions of the present invention.

In certain aspects, a nucleic acid comprises a wild-type or a mutantnucleic acid. In particular aspects, a nucleic acid encodes for orcomprises a transcribed nucleic acid. In other aspects, a nucleic acidcomprises a nucleic acid segment, or a biologically functionalequivalent thereof. In particular aspects, a nucleic acid encodes aprotein, polypeptide, peptide.

The term “nucleic acid” is well known in the art and may be usedinterchangeably herein with the term “polynucleotide.” A “nucleic acid”as used herein will generally refer to a molecule (i.e., a strand) ofDNA, RNA or a derivative or analog thereof, comprising a nucleobase. Anucleobase includes, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g., an adenine “A,” a guanine “G,” athymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” ora C). The term “nucleic acid” encompass the terms “oligonucleotide” and“polynucleotide,” each as a subgenus of the term “nucleic acid.” Theterm “oligonucleotide” refers to a molecule of between about 3 and about100 nucleobases in length. The term “polynucleotide” refers to at leastone molecule of greater than about 100 nucleobases in length.

These definitions generally refer to a single-stranded molecule, but inspecific embodiments will also encompass an additional strand that ispartially, substantially or fully complementary to the single-strandedmolecule. Thus, a nucleic acid may encompass a double-stranded moleculeor a triple-stranded molecule that comprises one or more complementarystrand(s) or “complement(s)” of a particular sequence comprising amolecule. As used herein, a single stranded nucleic acid may be denotedby the prefix “ss,” a double stranded nucleic acid by the prefix “ds,”and a triple stranded nucleic acid by the prefix “ts.”

A. Nucleobases

As used herein a “nucleobase” refers to a heterocyclic base, such as forexample a naturally occurring nucleobase (i.e., an A, T, G, C or U)found in at least one naturally occurring nucleic acid (i.e., DNA andRNA), and naturally or non-naturally occurring derivative(s) and analogsof such a nucleobase. A nucleobase generally can form one or morehydrogen bonds (“anneal” or “hybridize”) with at least one naturallyoccurring nucleobase in manner that may substitute for naturallyoccurring nucleobase pairing (e.g., the hydrogen bonding between A andT, G and C, and A and U).

“Purine” and/or “pyrimidine” nucleobase(s) encompass naturally occurringpurine and/or pyrimidine nucleobases and also derivative(s) andanalog(s) thereof, including but not limited to, those a purine orpyrimidine substituted by one or more of an alkyl, carboxyalkyl, amino,hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol oralkylthiol moeity. Preferred alkyl (e.g., alkyl, caboxyalkyl, etc.)moieties comprise of from about 1, about 2, about 3, about 4, about 5,to about 6 carbon atoms. Other non-limiting examples of a purine orpyrimidine include a deazapurine, a 2,6-diaminopurine, a 5-fluorouracil,a xanthine, a hypoxanthine, a 8-bromoguanine, a 8-chloroguanine, abromothymine, a 8-aminoguanine, a 8-hydroxyguanine, a 8-methylguanine, a8-thioguanine, an azaguanine, a 2-aminopurine, a 5-ethylcytosine, a5-methylcyosine, a 5-bromouracil, a 5-ethyluracil, a 5-iodouracil, a5-chlorouracil, a 5-propyluracil, a thiouracil, a 2-methyladenine, amethylthioadenine, a N,N-diemethyladenine, an azaadenines, a8-bromoadenine, a 8-hydroxyadenine, a 6-hydroxyaminopurine, a6-thiopurine, a 4-(6-aminohexyl/cytosine), and the like.

A nucleobase may be comprised in a nucleoside or nucleotide, using anychemical or natural synthesis method described herein or known to one ofordinary skill in the art.

B. Nucleosides

As used herein, a “nucleoside” refers to an individual chemical unitcomprising a nucleobase covalently attached to a nucleobase linkermoiety. A non-limiting example of a “nucleobase linker moiety” is asugar comprising 5-carbon atoms (i.e., a “5-carbon sugar”), includingbut not limited to a deoxyribose, a ribose, an arabinose, or aderivative or an analog of a 5-carbon sugar. Non-limiting examples of aderivative or an analog of a 5-carbon sugar include a2′-fluoro-2′-deoxyribose or a carbocyclic sugar where a carbon issubstituted for an oxygen atom in the sugar ring.

Different types of covalent attachment(s) of a nucleobase to anucleobase linker moiety are known in the art. By way of non-limitingexample, a nucleoside comprising a purine (i.e., A or G) or a7-deazapurine nucleobase typically covalently attaches the 9 position ofa purine or a 7-deazapurine to the 1′-position of a 5-carbon sugar. Inanother non-limiting example, a nucleoside comprising a pyrimidinenucleobase (i.e., C, T or U) typically covalently attaches a 1 positionof a pyrimidine to a 1′-position of a 5-carbon sugar (Kornberg andBaker, 1992).

C. Nucleotides

As used herein, a “nucleotide” refers to a nucleoside further comprisinga “backbone moiety”. A backbone moiety generally covalently attaches anucleotide to another molecule comprising a nucleotide, or to anothernucleotide to form a nucleic acid. The “backbone moiety” in naturallyoccurring nucleotides typically comprises a phosphorus moiety, which iscovalently attached to a 5-carbon sugar. The attachment of the backbonemoiety typically occurs at either the 3′- or 5′-position of the 5-carbonsugar. However, other types of attachments are known in the art,particularly when a nucleotide comprises derivatives or analogs of anaturally occurring 5-carbon sugar or phosphorus moiety.

D. Nucleic Acid Analogs

A nucleic acid may comprise, or be composed entirely of, a derivative oranalog of a nucleobase, a nucleobase linker moiety and/or backbonemoiety that may be present in a naturally occurring nucleic acid. Asused herein a “derivative” refers to a chemically modified or alteredform of a naturally occurring molecule, while the terms “mimic” or“analog” refer to a molecule that may or may not structurally resemble anaturally occurring molecule or moiety, but possesses similar functions.As used herein, a “moiety” generally refers to a smaller chemical ormolecular component of a larger chemical or molecular structure.Nucleobase, nucleoside and nucleotide analogs or derivatives are wellknown in the art, and have been described (see for example, Scheit,1980, incorporated herein by reference).

Additional non-limiting examples of nucleosides, nucleotides or nucleicacids comprising 5-carbon sugar and/or backbone moiety derivatives oranalogs, include those in U.S. Pat. No. 5,681,947 which describesoligonucleotides comprising purine derivatives that form triple helixeswith and/or prevent expression of dsDNA; U.S. Pat. Nos. 5,652,099 and5,763,167 which describe nucleic acids incorporating fluorescent analogsof nucleosides found in DNA or RNA, particularly for use as flourescentnucleic acids probes; U.S. Pat. No. 5,614,617 which describesoligonucleotide analogs with substitutions on pyrimidine rings thatpossess enhanced nuclease stability; U.S. Pat. Nos. 5,670,663, 5,872,232and 5,859,221 which describe oligonucleotide analogs with modified5-carbon sugars (i.e., modified 2′-deoxyfuranosyl moieties) used innucleic acid detection; U.S. Pat. No. 5,446,137 which describesoligonucleotides comprising at least one 5-carbon sugar moietysubstituted at the 4′ position with a substituent other than hydrogenthat can be used in hybridization assays; U.S. Pat. No. 5,886,165 whichdescribes oligonucleotides with both deoxyribonucleotides with 3′-5′internucleotide linkages and ribonucleotides with 2′-5′ internucleotidelinkages; U.S. Pat. No. 5,714,606 which describes a modifiedinternucleotide linkage wherein a 3′-position oxygen of theinternucleotide linkage is replaced by a carbon to enhance the nucleaseresistance of nucleic acids; U.S. Pat. No. 5,672,697 which describesoligonucleotides containing one or more 5′ methylene phosphonateinternucleotide linkages that enhance nuclease resistance; U.S. Pat.Nos. 5,466,786 and 5,792,847 which describe the linkage of a substituentmoeity which may comprise a drug or label to the 2′carbon of anoligonucleotide to provide enhanced nuclease stability and ability todeliver drugs or detection moieties; U.S. Pat. No. 5,223,618 whichdescribes oligonucleotide analogs with a 2 or 3 carbon backbone linkageattaching the 4′ position and 3′ position of adjacent 5-carbon sugarmoiety to enhanced cellular uptake, resistance to nucleases andhybridization to target RNA; U.S. Pat. No. 5,470,967 which describesoligonucleotides comprising at least one sulfamate or sulfamideinternucleotide linkage that are useful as nucleic acid hybridizationprobe; U.S. Pat. Nos. 5,378,825, 5,777,092, 5,623,070, 5,610,289 and5,602,240 which describe oligonucleotides with three or four atom linkermoeity replacing phosphodiester backbone moeity used for improvednuclease resistance, cellular uptake and regulating RNA expression; U.S.Pat. No. 5,858,988 which describes hydrophobic carrier agent attached tothe 2′-O position of oligonuceotides to enhanced their membranepermeability and stability; U.S. Pat. No. 5,214,136 which describesolignucleotides conjugaged to anthraquinone at the 5′ terminus thatpossess enhanced hybridization to DNA or RNA; enhanced stability tonucleases; U.S. Pat. No. 5,700,922 which describes PNA-DNA-PNA chimeraswherein the DNA comprises 2′-deoxy-erythropentofuranosyl nucleotides forenhanced nuclease resistance, binding affinity, and ability to activateRNase H; and U.S. Pat. No. 5,708,154 which describes RNA linked to a DNAto form a DNA-RNA hybrid.

E. Polyether and Peptide Nucleic Acids

In certain embodiments, it is contemplated that a nucleic acidcomprising a derivative or analog of a nucleoside or nucleotide may beused in the methods and compositions of the invention. A non-limitingexample is a “polyether nucleic acid”, described in U.S. Pat. No.5,908,845, incorporated herein by reference. In a polyether nucleicacid, one or more nucleobases are linked to chiral carbon atoms in apolyether backbone.

Another non-limiting example is a “peptide nucleic acid”, also known asa “PNA”, “peptide-based nucleic acid analog” or “PENAM”, described inU.S. Pat. Nos. 5,786,461, 5891,625, 5,773,571, 5,766,855, 5,736,336,5,719,262, 5,714,331, 5,539,082, and WO 92/20702, each of which isincorporated herein by reference. Peptide nucleic acids generally haveenhanced sequence specificity, binding properties, and resistance toenzymatic degradation in comparison to molecules such as DNA and RNA(Egholm et al., 1993; PCT/EP/01219). A peptide nucleic acid generallycomprises one or more nucleotides or nucleosides that comprise anucleobase moiety, a nucleobase linker moeity that is not a 5-carbonsugar, and/or a backbone moiety that is not a phosphate backbone moiety.Examples of nucleobase linker moieties described for PNAs include azanitrogen atoms, amido and/or ureido tethers (see for example, U.S. Pat.No. 5,539,082). Examples of backbone moieties described for PNAs includean aminoethylglycine, polyamide, polyethyl, polythioamide,polysulfinamide or polysulfonamide backbone moiety.

In certain embodiments, a nucleic acid analogue such as a peptidenucleic acid may be used to inhibit nucleic acid amplification, such asin PCR, to reduce false positives and discriminate between single basemutants, as described in U.S. Pat. No. 5,891,625. Other modificationsand uses of nucleic acid analogs are known in the art, and areencompassed by the gp36 polynucleotide. In a non-limiting example, U.S.Pat. No. 5,786,461 describes PNAs with amino acid side chains attachedto the PNA backbone to enhance solubility of the molecule. In anotherexample, the cellular uptake property of PNAs is increased by attachmentof a lipophilic group. U.S. application Ser. No. 117,363 describesseveral alkylamino moeities used to enhance cellular uptake of a PNA.Another example is described in U.S. Pat. Nos. 5,766,855, 5,719,262,5,714,331 and 5,736,336, which describe PNAs comprising naturally andnon-naturally occurring nucleobases and alkylamine side chains thatprovide improvements in sequence specificity, solubility and/or bindingaffinity relative to a naturally occurring nucleic acid.

F. Preparation of Nucleic Acids

A nucleic acid may be made by any technique known to one of ordinaryskill in the art, such as for example, chemical synthesis, enzymaticproduction or biological production. Non-limiting examples of asynthetic nucleic acid (e.g., a synthetic oligonucleotide), include anucleic acid made by in vitro chemically synthesis usingphosphotriester, phosphite or phosphoramidite chemistry and solid phasetechniques such as described in EP 266,032, incorporated herein byreference, or via deoxynucleoside H-phosphonate intermediates asdescribed by Froehler et al., 1986 and U.S. Pat. No. 5,705,629, eachincorporated herein by reference. In the methods of the presentinvention, one or more oligonucleotide may be used. Various differentmechanisms of oligonucleotide synthesis have been disclosed in forexample, U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566,4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which isincorporated herein by reference.

A non-limiting example of an enzymatically produced nucleic acid includeone produced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of anoligonucleotide described in U.S. Pat. No. 5,645,897, incorporatedherein by reference. A non-limiting example, of a biologically producednucleic acid includes a recombinant nucleic acid produced (i.e.,replicated) in a living cell, such as a recombinant DNA vectorreplicated in bacteria (see for example, Sambrook et al. 1989,incorporated herein by reference).

G. Purification of Nucleic Acids

A nucleic acid may be purified on polyacrylamide gels, cesium chloridecentrifugation gradients, or by any other means known to one of ordinaryskill in the art (see for example, Sambrook et al., 1989, incorporatedherein by reference).

In certain aspect, the present invention concerns a nucleic acid that isan isolated nucleic acid. As used herein, the term “isolated nucleicacid” refers to a nucleic acid molecule (e.g., an RNA or DNA molecule)that has been isolated free of, or is otherwise free of, the bulk of thetotal genomic and transcribed nucleic acids of one or more cells. Incertain embodiments, “isolated nucleic acid” refers to a nucleic acidthat has been isolated free of, or is otherwise free of, bulk ofcellular components or in vitro reaction components such as for example,macromolecules such as lipids or proteins, small biological molecules,and the like.

H. Nucleic Acid Segments

In certain embodiments, the nucleic acid is a nucleic acid segment. Asused herein, the term “nucleic acid segment,” are smaller fragments of anucleic acid, such as for non-limiting example, those that encode onlypart of the peptide or polypeptide sequence. Thus, a “nucleic acidsegment” may comprise any part of a gene sequence, of from about 2nucleotides to the full length of the peptide or polypeptide encodingregion.

Various nucleic acid segments may be designed based on a particularnucleic acid sequence, and may be of any length. By assigning numericvalues to a sequence, for example, the first residue is 1, the secondresidue is 2, etc., an algorithm defining all nucleic acid segments canbe generated:

n to n+y

where n is an integer from 1 to the last number of the sequence and y isthe length of the nucleic acid segment minus one, where n+y does notexceed the last number of the sequence. Thus, for a 10 mer, the nucleicacid segments correspond to bases 1 to 10, 2 to 11, 3 to 12 . . . and soon. For a 15-mer, the nucleic acid segments correspond to bases 1 to 15,2 to 16, 3 to 17 . . . and so on. For a 20-mer, the nucleic segmentscorrespond to bases 1 to 20, 2 to 21, 3 to 22 . . . and so on. Incertain embodiments, the nucleic acid segment may be a probe or primer.As used herein, a “probe” generally refers to a nucleic acid used in adetection method or composition. As used herein, a “primer” generallyrefers to a nucleic acid used in an extension or amplification method orcomposition.

I. Nucleic Acid Complements

The present invention also encompasses a nucleic acid that iscomplementary to one or more other nucleic acids. In specificembodiments, for example, a nucleic acid is employed for antisense orsiRNA purposes, such as to inhibit at least partially expression of apolynucleotide.

In particular embodiments the invention encompasses a nucleic acid or anucleic acid segment complementary to the sequence set forth herein, forexample. A nucleic acid is “complement(s)” or is “complementary” toanother nucleic acid when it is capable of base-pairing with anothernucleic acid according to the standard Watson-Crick, Hoogsteen orreverse Hoogsteen binding complementarity rules. As used herein “anothernucleic acid” may refer to a separate molecule or a spatial separatedsequence of the same molecule.

As used herein, the term “complementary” or “complement(s)” also refersto a nucleic acid comprising a sequence of consecutive nucleobases orsemiconsecutive nucleobases (e.g., one or more nucleobase moieties arenot present in the molecule) capable of hybridizing to another nucleicacid strand or duplex even if less than all the nucleobases do not basepair with a counterpart nucleobase. In certain embodiments, a“complementary” nucleic acid comprises a sequence in which about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%,about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, to about 100%, and any rangederivable therein, of the nucleobase sequence is capable of base-pairingwith a single or double stranded nucleic acid molecule duringhybridization. In certain embodiments, the term “complementary” refersto a nucleic acid that may hybridize to another nucleic acid strand orduplex in stringent conditions, as would be understood by one ofordinary skill in the art.

In certain embodiments, a “partly complementary” nucleic acid comprisesa sequence that may hybridize in low stringency conditions to a singleor double stranded nucleic acid, or contains a sequence in which lessthan about 70% of the nucleobase sequence is capable of base-pairingwith a single or double stranded nucleic acid molecule duringhybridization.

J. Hybridization

As used herein, “hybridization”, “hybridizes” or “capable ofhybridizing” is understood to mean the forming of a double or triplestranded molecule or a molecule with partial double or triple strandednature. The term “anneal” as used herein is synonymous with “hybridize.”The term “hybridization”, “hybridize(s)” or “capable of hybridizing”encompasses the terms “stringent condition(s)” or “high stringency” andthe terms “low stringency” or “low stringency condition(s).”

As used herein “stringent condition(s)” or “high stringency” are thoseconditions that allow hybridization between or within one or morenucleic acid strand(s) containing complementary sequence(s), butprecludes hybridization of random sequences. Stringent conditionstolerate little, if any, mismatch between a nucleic acid and a targetstrand. Such conditions are well known to those of ordinary skill in theart, and are preferred for applications requiring high selectivity.Non-limiting applications include isolating a nucleic acid, such as agene or a nucleic acid segment thereof, or detecting at least onespecific mRNA transcript or a nucleic acid segment thereof, and thelike.

Stringent conditions may comprise low salt and/or high temperatureconditions, such as provided by about 0.02 M to about 0.15 M NaCl, forexample, at temperatures of about 50° C. to about 70° C. or, forexample, wherein said stringent conditions are hybridization at 50-65°C., 5×SSPC, 50% formamide; wash 50-65° C., 5×SSPC; or wash at 60° C.,0.5×SSC, 0.1% SDS. It is understood that the temperature and ionicstrength of a desired stringency are determined in part by the length ofthe particular nucleic acid(s), the length and nucleobase content of thetarget sequence(s), the charge composition of the nucleic acid(s), andto the presence or concentration of formamide, tetramethylammoniumchloride or other solvent(s) in a hybridization mixture.

It is also understood that these ranges, compositions and conditions forhybridization are mentioned by way of non-limiting examples only, andthat the desired stringency for a particular hybridization reaction isoften determined empirically by comparison to one or more positive ornegative controls. Depending on the application envisioned it ispreferred to employ varying conditions of hybridization to achievevarying degrees of selectivity of a nucleic acid towards a targetsequence. In a non-limiting example, identification or isolation of arelated target nucleic acid that does not hybridize to a nucleic acidunder stringent conditions may be achieved by hybridization at lowtemperature and/or high ionic strength. Such conditions are termed “lowstringency” or “low stringency conditions”, and non-limiting examples oflow stringency include hybridization performed at about 0.15 M to about0.9 M NaCl at a temperature range of about 20° C. to about 50° C. Ofcourse, it is within the skill of one in the art to further modify thelow or high stringency conditions to suite a particular application.

V. Nucleic Acid-Based Expression Systems

In particular embodiments, the present invention concerns apolynucleotide that encodes an immunoreactive ehrlichiae polypeptide,and also includes delivering the polynucleotide encoding thepolypeptide, or encoded product thereof, to an individual in needthereof, such as an individual infected with Erhlichia and/or anindividual susceptible to being infected with Erhlichia. For the sake ofbrevity, the following section will refer to any E. canis gp19 nucleicacid compositions and/or nucleic acid-based expression system of thepresent invention.

The present invention is directed toward substantially pure and/orisolated DNA sequence encoding an immunoreactive Ehrlichia composition.Generally, the encoded protein comprises an N-terminal sequence, whichmay be cleaved after post-translational modification resulting in theproduction of mature protein.

It is well-known in the art that because of the degeneracy of thegenetic code (i.e., for most amino acids, more than one nucleotidetriplet (codon) codes for a single amino acid), different nucleotidesequences can code for a particular amino acid, or polypeptide. Thus,the polynucleotide sequences of the subject invention include any of theprovided exemplary sequences or a degenerate variant of such a sequence,for example. In particular aspects of the invention, a degeneratevariant comprises a sequence that is not identical to a sequence of theinvention but that still retains one or more properties of a sequence ofthe invention.

As used herein, “substantially pure DNA” means DNA that is not part of amilieu in which the DNA naturally occurs, by virtue of separation(partial or total purification) of some or all of the molecules of thatmilieu, or by virtue of alteration of sequences that flank the claimedDNA. The term therefore includes, for example, a recombinant DNA whichis incorporated into a vector, into an autonomously replicating plasmidor virus, or into the genomic DNA of a prokaryote or eukaryote; or thatexists as a separate molecule (e.g., a cDNA or a genomic or cDNAfragment produced by polymerase chain reaction (PCR) or restrictionendonuclease digestion) independent of other sequences. It also includesa recombinant DNA, which is part of a hybrid gene encoding additionalpolypeptide sequence, e.g., a fusion protein.

The present invention is further directed to an expression vectorcomprising a polynucleotide encoding an immunoreactive Ehrlichiaecomposition and capable of expressing the polynucleotide when the vectoris introduced into a cell. In specific embodiments, the vector comprisesin operable linkage the following: a) an origin of replication; b) apromoter; and c) a DNA sequence coding for the protein.

As used herein “vector” may be defined as a replicable nucleic acidconstruct, e.g., a plasmid or viral nucleic acid. Vectors may be used toamplify and/or express nucleic acid encoding an immunoreactivecomposition of Ehrlichiae. An expression vector is a replicableconstruct in which a nucleic acid sequence encoding a polypeptide isoperably linked to suitable control sequences capable of effectingexpression of the polypeptide in a cell. The need for such controlsequences will vary depending upon the cell selected and thetransformation method chosen. Generally, control sequences include atranscriptional promoter and/or enhancer, suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation, for example. Methods that are well-knownto those skilled in the art can be used to construct expression vectorscomprising appropriate transcriptional and translational controlsignals. See for example, the techniques described in Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold SpringHarbor Press, N.Y. A polynucleotide sequence to be expressed and itstranscription control sequences are defined as being “operably linked”if the transcription control sequences effectively control thetranscription of the polynucleotide sequence. Vectors of the inventioninclude, but are not limited to, plasmid vectors and viral vectors.Preferred viral vectors of the invention are those derived fromretroviruses, adenovirus, adeno-associated virus, SV40 virus, or herpesviruses, for example.

In general, expression vectors comprise promoter sequences thatfacilitate the efficient transcription of the polynucleotide to beexpressed, are used in connection with a host cell. As used herein, theterm “host” is meant to include not only prokaryotes but alsoeukaryotes, such as yeast, plant and animal cells. A recombinantpolynucleotide that encodes an immunoreactive composition of Ehrlichiaeof the present invention can be used to transform a host using any ofthe techniques commonly known to those of ordinary skill in the art.Prokaryotic hosts may include E. coli, S. tymphimurium, Serratiamarcescens and Bacillus subtilis. Eukaryotic hosts include yeasts, suchas Pichia pastoris, mammalian cells and insect cells.

The following description concerns exemplary elements, reagents, andmethods for polynucleotides and nucleic acid delivery of an Ehrlichiapolynucleotide.

A. Vectors

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques (see, for example, Maniatis et al., 1988 and Ausubel et al.,1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for a RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

1. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as, for example, thepromoter for the mammalian terminal deoxynucleotidyl transferase geneand the promoter for the SV40 late genes, a discrete element overlyingthe start site itself helps to fix the place of initiation. Additionalpromoter elements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30 110 bp upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include the betalactamase (penicillinase), lactose and tryptophan (trp) promotersystems. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202 and 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in the cell,organelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al., 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

The promoter may be one suitable for use in a prokaryotic cell, aeukaryotic cell, or both. Additionally any promoter/enhancer combination(as per, for example, the Eukaryotic Promoter Data Base EPDB) could alsobe used to drive expression. Use of a T3, T7 or SP6 cytoplasmicexpression system is one possible embodiment.

2. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, each herein incorporated by reference).

3. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector (see, for example, Carbonelli et al., 1999, Levensonet al., 1998, and Cocea, 1997, incorporated herein by reference.)“Restriction enzyme digestion” refers to catalytic cleavage of a nucleicacid molecule with an enzyme that functions only at specific locationsin a nucleic acid molecule. Many of these restriction enzymes arecommercially available. Use of such enzymes is widely understood bythose of skill in the art. Frequently, a vector is linearized orfragmented using a restriction enzyme that cuts within the MCS to enableexogenous sequences to be ligated to the vector. “Ligation” refers tothe process of forming phosphodiester bonds between two nucleic acidfragments, which may or may not be contiguous with each other.Techniques involving restriction enzymes and ligation reactions are wellknown to those of skill in the art of recombinant technology.

4. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (see,for example, Chandler et al., 1997, herein incorporated by reference.)

5. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

6. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal or the bovine growth hormone polyadenylationsignal, convenient and known to function well in various target cells.Polyadenylation may increase the stability of the transcript or mayfacilitate cytoplasmic transport.

7. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

8. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

9. Plasmid Vectors

In certain embodiments, a plasmid vector is contemplated for use totransform a host cell. In general, plasmid vectors containing repliconand control sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries a replication site, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. In anon-limiting example, E. coli is often transformed using derivatives ofpBR322, a plasmid derived from an E. coli species. pBR322 contains genesfor ampicillin and tetracycline resistance and thus provides easy meansfor identifying transformed cells. The pBR plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, forexample, promoters which can be used by the microbial organism forexpression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example, thephage lambda GEMTM 11 may be utilized in making a recombinant phagevector which can be used to transform host cells, such as, for example,E. coli LE392.

Further useful plasmid vectors include pIN vectors (Inouye et al.,1985); and pGEX vectors, for use in generating glutathione S transferase(GST) soluble fusion proteins for later purification and separation orcleavage. Other suitable fusion proteins are those with betagalactosidase, ubiquitin, and the like.

Bacterial host cells, for example, E. coli, comprising the expressionvector are grown in any of a number of suitable media, for example, LB.The expression of the recombinant protein in certain vectors may beinduced, as would be understood by those of skill in the art, bycontacting a host cell with an agent specific for certain promoters,e.g., by adding IPTG to the media or by switching incubation to a highertemperature. After culturing the bacteria for a further period,generally of between 2 and 24 h, the cells are collected bycentrifugation and washed to remove residual media.

10. Viral Vectors

The ability of certain viruses to infect cells or enter cells viareceptor mediated endocytosis, and to integrate into host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g.,mammalian cells). Components of the present invention may comprise aviral vector that encode one or more compositions or other componentssuch as, for example, an immunomodulator or adjuvant. Non-limitingexamples of virus vectors that may be used to deliver a nucleic acid ofthe present invention are described below.

a. Adenoviral Vectors

A particular method for delivery of the nucleic acid involves the use ofan adenovirus expression vector. Although adenovirus vectors are knownto have a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to ultimately express a tissue orcell specific construct that has been cloned therein. Knowledge of thegenetic organization or adenovirus, a 36 kb, linear, double stranded DNAvirus, allows substitution of large pieces of adenoviral DNA withforeign sequences up to 7 kb (Grunhaus and Horwitz, 1992).

b. AAV Vectors

The nucleic acid may be introduced into the cell using adenovirusassisted transfection. Increased transfection efficiencies have beenreported in cell systems using adenovirus coupled systems (Kelleher andVos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno associated virus(AAV) is an attractive vector system for use in the compositions of thepresent invention as it has a high frequency of integration and it caninfect nondividing cells, thus making it useful for delivery of genesinto mammalian cells, for example, in tissue culture (Muzyczka, 1992) orin vivo. AAV has a broad host range for infectivity (Tratschin et al.,1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al.,1988). Details concerning the generation and use of rAAV vectors aredescribed in U.S. Pat. Nos. 5,139,941 and 4,797,368, each incorporatedherein by reference.

c. Retroviral Vectors

Retroviruses have useful as delivery vectors due to their ability tointegrate their, genes into the host genome, transferring a large amountof foreign genetic material, infecting a broad spectrum of species andcell types and of being packaged in special cell lines (Miller, 1992).

In order to construct a retroviral vector, a nucleic acid (e.g., oneencoding a composition of interest) is inserted into the viral genome inthe place of certain viral sequences to produce a virus that isreplication defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into a special cell line (e.g., bycalcium phosphate precipitation for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media (Nicolasand Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are able toinfect a broad variety of cell types. However, integration and stableexpression require the division of host cells (Paskind et al., 1975).

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Lentiviral vectors are well known in the art(see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomeret al., 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples oflentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 andthe Simian Immunodeficiency Virus: SIV. Lentiviral vectors have beengenerated by multiply attenuating the HIV virulence genes, for example,the genes env, vif, vpr, vpu and nef are deleted making the vectorbiologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. For example, recombinantlentivirus capable of infecting a non-dividing cell wherein a suitablehost cell is transfected with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat is describedin U.S. Pat. No. 5,994,136, incorporated herein by reference. One maytarget the recombinant virus by linkage of the envelope protein with anantibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including a regulatoryregion) of interest into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target-specific.

d. Other Viral Vectors

Other viral vectors may be employed as vaccine constructs in the presentinvention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988),sindbis virus, cytomegalovirus and herpes simplex virus may be employed.They offer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

e. Delivery Using Modified Viruses

A nucleic acid to be delivered may be housed within an infective virusthat has been engineered to express a specific binding ligand. The virusparticle will thus bind specifically to the cognate receptors of thetarget cell and deliver the contents to the cell. A novel approachdesigned to allow specific targeting of retrovirus vectors was developedbased on the chemical modification of a retrovirus by the chemicaladdition of lactose residues to the viral envelope. This modificationcan permit the specific infection of hepatocytes via sialoglycoproteinreceptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et al.,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux et al., 1989).

11. Vector Delivery and Cell Transformation

Suitable methods for ehrlichial nucleic acid delivery for transformationof an organelle, a cell, a tissue or an organism for use with thecurrent invention are believed to include virtually any method by whicha nucleic acid (e.g., DNA) can be introduced into an organelle, a cell,a tissue or an organism, as described herein or as would be known to oneof ordinary skill in the art. Such methods include, but are not limitedto, direct delivery of DNA such as by ex vivo transfection (Wilson etal., 1989, Nabel et al, 1989), by injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),including microinjection (Harlan and Weintraub, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S.Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al.,1986; Potter et al., 1984); by calcium phosphate precipitation (Grahamand Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); byusing DEAE dextran followed by polyethylene glycol (Gopal, 1985); bydirect sonic loading (Fechheimer et al., 1987); by liposome mediatedtransfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau etal., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991)and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988);by microprojectile bombardment (PCT Application Nos. WO 94/09699 and95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318,5,538,877 and 5,538,880, and each incorporated herein by reference); byagitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat.Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); byAgrobacterium mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); by PEG mediatedtransformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition mediated DNA uptake (Potrykus et al., 1985), andany combination of such methods. Through the application of techniquessuch as these, organelle(s), cell(s), tissue(s) or organism(s) may bestably or transiently transformed.

a. Ex Vivo Transformation

Methods for tranfecting vascular cells and tissues removed from anorganism in an ex vivo setting are known to those of skill in the art.For example; cannine endothelial cells have been genetically altered byretrovial gene tranfer in vitro and transplanted into a canine (Wilsonet al., 1989). In another example, yucatan minipig endothelial cellswere tranfected by retrovirus in vitro and transplated into an arteryusing a double-ballonw catheter (Nabel et al., 1989). Thus, it iscontemplated that cells or tissues may be removed and tranfected ex vivousing the nucleic acids of the present invention. In particular aspects,the transplanted cells or tissues may be placed into an organism. Inpreferred facets, a nucleic acid is expressed in the transplated cellsor tissues.

b. Injection

In certain embodiments, a nucleic acid may be delivered to an organelle,a cell, a tissue or an organism via one or more injections (i.e., aneedle injection), such as, for example, subcutaneously, intradermally,intramuscularly, intervenously, intraperitoneally, etc. Methods ofinjection of vaccines are well known to those of ordinary skill in theart (e.g., injection of a composition comprising a saline solution).Further embodiments of the present invention include the introduction ofa nucleic acid by direct microinjection. Direct microinjection has beenused to introduce nucleic acid constructs into Xenopus oocytes (Harlandand Weintraub, 1985). The amount of composition used may vary upon thenature of the antigen as well as the organelle, cell, tissue or organismused

c. Electroporation

In certain embodiments of the present invention, a nucleic acid isintroduced into an organelle, a cell, a tissue or an organism viaelectroporation. Electroporation involves the exposure of a suspensionof cells and DNA to a high voltage electric discharge. In some variantsof this method, certain cell wall degrading enzymes, such as pectindegrading enzymes, are employed to render the target recipient cellsmore susceptible to transformation by electroporation than untreatedcells (U.S. Pat. No. 5,384,253, incorporated herein by reference).Alternatively, recipient cells can be made more susceptible totransformation by mechanical wounding.

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre B lymphocytes have been transfected with humankappa immunoglobulin genes (Potter et al., 1984), and rat hepatocyteshave been transfected with the chloramphenicol acetyltransferase gene(Tur Kaspa et al., 1986) in this manner.

To effect transformation by electroporation in cells such as, forexample, plant cells, one may employ either friable tissues, such as asuspension culture of cells or embryogenic callus or alternatively onemay transform immature embryos or other organized tissue directly. Inthis technique, one would partially degrade the cell walls of the chosencells by exposing them to pectin degrading enzymes (pectolyases) ormechanically wounding in a controlled manner. Examples of some specieswhich have been transformed by electroporation of intact cells includemaize (U.S. Pat. No. 5,384,253; Rhodes et al., 1995; D'Halluin et al.,1992), wheat (Zhou et al., 1993), tomato (Hou and Lin, 1996), soybean(Christou et al., 1987) and tobacco (Lee et al., 1989).

One also may employ protoplasts for electroporation transformation ofplant cells (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon derivedprotoplasts is described by Dhir and Widholm in International PatentApplication No. WO 9217598, incorporated herein by reference. Otherexamples of species for which protoplast transformation has beendescribed include barley (Lazerri, 1995), sorghum (Battraw et al.,1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) andtomato (Tsukada, 1989).

d. Calcium Phosphate

In other embodiments of the present invention, a nucleic acid isintroduced to the cells using calcium phosphate precipitation. Human KBcells have been transfected with adenovirus 5 DNA (Graham and Van DerEb, 1973) using this technique. Also in this manner, mouse L(A9), mouseC127, CHO, CV 1, BHK, NIH3T3 and HeLa cells were transfected with aneomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes weretransfected with a variety of marker genes (Rippe et al., 1990).

e. DEAE Dextran

In another embodiment, a nucleic acid is delivered into a cell usingDEAE dextran followed by polyethylene glycol. In this manner, reporterplasmids were introduced into mouse myeloma and erythroleukemia cells(Gopal, 1985).

f. Sonication Loading

Additional embodiments of the present invention include the introductionof a nucleic acid by direct sonic loading. LTK fibroblasts have beentransfected with the thymidine kinase gene by sonication loading(Fechheimer et al., 1987).

g. Liposome-Mediated Transfection

In a further embodiment of the invention, an ehrlichial nucleic acid maybe comprised with a lipid complex such as, for example, comprised in aliposome. Liposomes are vesicular structures characterized by aphospholipid bilayer membrane and an inner aqueous medium. Multilamellarliposomes have multiple lipid layers separated by aqueous medium. Theyform spontaneously when phospholipids are suspended in an excess ofaqueous solution. The lipid components undergo self rearrangement beforethe formation of closed structures and entrap water and dissolvedsolutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Alsocontemplated is an nucleic acid complexed with Lipofectamine (Gibco BRL)or Superfect (Qiagen).

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful (Nicolau and Sene, 1982; Fraley et al.,1979; Nicolau et al., 1987). The feasibility of liposome mediateddelivery and expression of foreign DNA in cultured chick embryo, HeLaand hepatoma cells has also been demonstrated (Wong et al., 1980).

In certain embodiments of the invention, a liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry of liposomeencapsulated DNA (Kaneda et al., 1989). In other embodiments, a liposomemay be complexed or employed in conjunction with nuclear non histonechromosomal proteins (HMG 1) (Kato et al., 1991). In yet furtherembodiments, a liposome may be complexed or employed in conjunction withboth HVJ and HMG 1. In other embodiments, a delivery vehicle maycomprise a ligand and a liposome.

h. Receptor-Mediated Transfection

Still further, a nucleic acid may be delivered to a target cell viareceptor mediated delivery vehicles. These take advantage of theselective uptake of macromolecules by receptor-mediated endocytosis thatwill be occurring in a target cell. In view of the cell type specificdistribution of various receptors, this delivery method adds anotherdegree of specificity to the present invention.

Certain receptor mediated gene targeting vehicles comprise a cellreceptor specific ligand and a nucleic acid binding agent. Otherscomprise a cell receptor specific ligand to which the nucleic acid to bedelivered has been operatively attached. Several ligands have been usedfor receptor mediated gene transfer (Wu and Wu, 1987; Wagner et al.,1990; Perales et al., 1994; Myers, EPO 0273085), which establishes theoperability of the technique. Specific delivery in the context ofanother mammalian cell type has been described (Wu and Wu, 1993;incorporated herein by reference). In certain aspects of the presentinvention, a ligand will be chosen to correspond to a receptorspecifically expressed on the target cell population.

In other embodiments, a nucleic acid delivery vehicle component of acell specific nucleic acid targeting vehicle may comprise a specificbinding ligand in combination with a liposome. The nucleic acid(s) to bedelivered are housed within the liposome and the specific binding ligandis functionally incorporated into the liposome membrane. The liposomewill thus specifically bind to the receptor(s) of a target cell anddeliver the contents to a cell. Such systems have been shown to befunctional using systems in which, for example, epidermal growth factor(EGF) is used in the receptor mediated delivery of a nucleic acid tocells that exhibit upregulation of the EGF receptor.

In still further embodiments, the nucleic acid delivery vehiclecomponent of a targeted delivery vehicle may be a liposome itself, whichwill preferably comprise one or more lipids or glycoproteins that directcell specific binding. For example, lactosyl ceramide, a galactoseterminal asialganglioside, have been incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes(Nicolau et al., 1987). It is contemplated that the tissue specifictransforming constructs of the present invention can be specificallydelivered into a target cell in a similar manner.

i. Microprojectile Bombardment

Microprojectile bombardment techniques can be used to introduce anucleic acid into at least one, organelle, cell, tissue or organism(U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No.5,610,042; and PCT Application WO 94/09699; each of which isincorporated herein by reference). This method depends on the ability toaccelerate DNA coated microprojectiles to a high velocity allowing themto pierce cell membranes and enter cells without killing them (Klein etal., 1987). There are a wide variety of microprojectile bombardmenttechniques known in the art, many of which are applicable to theinvention.

Microprojectile bombardment may be used to transform various cell(s),tissue(s) or organism(s), such as for example any plant species.Examples of species which have been transformed by microprojectilebombardment include monocot species such as maize (PCT Application WO95/06128), barley (Ritala et al., 1994; Hensgens et al., 1993), wheat(U.S. Pat. No. 5,563,055, incorporated herein by reference), rice(Hensgens et al., 1993), oat (Torbet et al., 1995; Torbet et al., 1998),rye (Hensgens et al., 1993), sugarcane (Bower et al., 1992), and sorghum(Casas et al., 1993; Hagio et al., 1991); as well as a number of dicotsincluding tobacco (Tomes et al., 1990; Buising and Benbow, 1994),soybean (U.S. Pat. No. 5,322,783, incorporated herein by reference),sunflower (Knittel et al. 1994), peanut (Singsit et al., 1997), cotton(McCabe and Martinell, 1993), tomato (VanEck et al. 1995), and legumesin general (U.S. Pat. No. 5,563,055, incorporated herein by reference).

In this microprojectile bombardment, one or more particles may be coatedwith at least one nucleic acid and delivered into cells by a propellingforce. Several devices for accelerating small particles have beendeveloped. One such device relies on a high voltage discharge togenerate an electrical current, which in turn provides the motive force(Yang et al., 1990). The microprojectiles used have consisted ofbiologically inert substances such as tungsten or gold particles orbeads. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. It is contemplated that in someinstances DNA precipitation onto metal particles would not be necessaryfor DNA delivery to a recipient cell using microprojectile bombardment.However, it is contemplated that particles may contain DNA rather thanbe coated with DNA. DNA coated particles may increase the level of DNAdelivery via particle bombardment but are not, in and of themselves,necessary.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells may be arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate.

An illustrative embodiment of a method for delivering DNA into a cell(e.g., a plant cell) by acceleration is the Biolistics Particle DeliverySystem, which can be used to propel particles coated with DNA or cellsthrough a screen, such as a stainless steel or Nytex screen, onto afilter surface covered with cells, such as for example, a monocot plantcells cultured in suspension. The screen disperses the particles so thatthey are not delivered to the recipient cells in large aggregates. It isbelieved that a screen intervening between the projectile apparatus andthe cells to be bombarded reduces the size of projectiles aggregate andmay contribute to a higher frequency of transformation by reducing thedamage inflicted on the recipient cells by projectiles that are toolarge.

12. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organism that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny. Asused herein, the terms “engineered” and “recombinant” cells or hostcells are intended to refer to a cell into which an exogenous nucleicacid sequence, such as, for example, a vector, has been introduced.Therefore, recombinant cells are distinguishable from naturallyoccurring cells which do not contain a recombinantly introduced nucleicacid.

In certain embodiments, it is contemplated that RNAs or proteinaceoussequences may be co-expressed with other selected RNAs or proteinaceoussequences in the same host cell. Co-expression may be achieved byco-transfecting the host cell with two or more distinct recombinantvectors. Alternatively, a single recombinant vector may be constructedto include multiple distinct coding regions for RNAs, which could thenbe expressed in host cells transfected with the single vector.

A tissue may comprise a host cell or cells to be transformed with acomposition of the invention. The tissue may be part or separated froman organism. In certain embodiments, a tissue may comprise, but is notlimited to, adipocytes, alveolar, ameloblasts, axon, basal cells, blood(e.g., lymphocytes), blood vessel, bone, bone marrow, brain, breast,cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial,epithelial, esophagus, facia, fibroblast, follicular, ganglion cells,glial cells, goblet cells, kidney, liver, lung, lymph node, muscle,neuron, ovaries, pancreas, peripheral blood, prostate, skin, skin, smallintestine, spleen, stem cells, stomach, testes, anthers, ascite tissue,cobs, ears, flowers, husks, kernels, leaves, meristematic cells, pollen,root tips, roots, silk, stalks, and all cancers thereof.

In certain embodiments, the host cell or tissue may be comprised in atleast one organism. In certain embodiments, the organism may be, but isnot limited to, a prokayote (e.g., a eubacteria, an archaea) or aneukaryote, as would be understood by one of ordinary skill in the art(see, for example, webpagehttp://phylogeny.arizona.edukree/phylogeny.html).

Numerous cell lines and cultures are available for use as a host cell,and they can be obtained through the American Type Culture Collection(ATCC), which is an organization that serves as an archive for livingcultures and genetic materials (www.atcc.org). An appropriate host canbe determined by one of skill in the art based on the vector backboneand the desired result. A plasmid or cosmid, for example, can beintroduced into a prokaryote host cell for replication of many vectors.Cell types available for vector replication and/or expression include,but are not limited to, bacteria, such as E. coli (e.g., E. coli strainRR1, E. coli LE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as wellas E. coli W3110 (F, lambda, prototrophic, ATCC No. 273325), DH5α,JM109, and KC8, bacilli such as Bacillus subtilis; and otherenterobacteriaceae such as Salmonella typhimurium, Serratia marcescens,various Pseudomonas specie, as well as a number of commerciallyavailable bacterial hosts such as SURE® Competent Cells and SOLOPACKGold Cells (STRATAGENE®, La Jolla). In certain embodiments, bacterialcells such as E. coli LE392 are particularly contemplated as host cellsfor phage viruses.

Examples of eukaryotic host cells for replication and/or expression of avector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, Cos,CHO, Saos, and PC12. Many host cells from various cell types andorganisms are available and would be known to one of skill in the art.Similarly, a viral vector may be used in conjunction with either aeukaryotic or prokaryotic host cell, particularly one that is permissivefor replication or expression of the vector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

13. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

Other examples of expression systems include STRATAGENE®'s COMPLETECONTROL Inducible Mammalian Expression System, which involves asynthetic ecdysone-inducible receptor, or its pET Expression System, anE. coli expression system. Another example of an inducible expressionsystem is available from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

It is contemplated that the proteins, polypeptides or peptides producedby the methods of the invention may be “overexpressed”, i.e., expressedin increased levels relative to its natural expression in cells. Suchoverexpression may be assessed by a variety of methods, including radiolabeling and/or protein purification. However, simple and direct methodsare preferred, for example, those involving SDS/PAGE and proteinstaining or western blotting, followed by quantitative analyses, such asdensitometric scanning of the resultant gel or blot. A specific increasein the level of the recombinant protein, polypeptide or peptide incomparison to the level in natural cells is indicative ofoverexpression, as is a relative abundance of the specific protein,polypeptides or peptides in relation to the other proteins produced bythe host cell and, e.g., visible on a gel.

In some embodiments, the expressed proteinaceous sequence forms aninclusion body in the host cell, the host cells are lysed, for example,by disruption in a cell homogenizer, washed and/or centrifuged toseparate the dense inclusion bodies and cell membranes from the solublecell components. This centrifugation can be performed under conditionswhereby the dense inclusion bodies are selectively enriched byincorporation of sugars, such as sucrose, into the buffer andcentrifugation at a selective speed. Inclusion bodies may be solubilizedin solutions containing high concentrations of urea (e.g. 8M) orchaotropic agents such as guanidine hydrochloride in the presence ofreducing agents, such as beta mercaptoethanol or DTT (dithiothreitol),and refolded into a more desirable conformation, as would be known toone of ordinary skill in the art.

VI. Biological Functional Equivalents

As modifications and/or changes may be made in the structure of thepolynucleotides and/or proteins according to the present invention,while obtaining molecules having similar or improved characteristics,such biologically functional equivalents are also encompassed within thepresent invention.

A. Modified Polynucleotides and Polypeptides

The biological functional equivalent may comprise a polynucleotide thathas been engineered to contain distinct sequences while at the same timeretaining the capacity to encode the “wild-type” or standard protein.This can be accomplished to the degeneracy of the genetic code, i.e.,the presence of multiple codons, which encode for the same amino acids.In one example, one of skill in the art may wish to introduce arestriction enzyme recognition sequence into a polynucleotide while notdisturbing the ability of that polynucleotide to encode a protein.

In another example, a polynucleotide made be (and encode) a biologicalfunctional equivalent with more significant changes. Certain amino acidsmay be substituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies, binding sites onsubstrate molecules, receptors, and such like. So-called “conservative”changes do not disrupt the biological activity of the protein, as thestructural change is not one that impinges of the protein's ability tocarry out its designed function. It is thus contemplated by theinventors that various changes may be made in the sequence of genes andproteins disclosed herein, while still fulfilling the goals of thepresent invention.

In terms of functional equivalents, it is well understood by the skilledartisan that, inherent in the definition of a “biologically functionalequivalent” protein and/or polynucleotide, is the concept that there isa limit to the number of changes that may be made within a definedportion of the molecule while retaining a molecule with an acceptablelevel of equivalent biological activity. Biologically functionalequivalents are thus defined herein as those proteins (andpolynucleotides) in selected amino acids (or codons) may be substituted.Functional activity.

In general, the shorter the length of the molecule, the fewer changesthat can be made within the molecule while retaining function. Longerdomains may have an intermediate number of changes. The full-lengthprotein will have the most tolerance for a larger number of changes.However, it must be appreciated that certain molecules or domains thatare highly dependent upon their structure may tolerate little or nomodification.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and/or the like. Ananalysis of the size, shape and/or type of the amino acid side-chainsubstituents reveals that arginine, lysine and/or histidine are allpositively charged residues; that alanine, glycine and/or serine are alla similar size; and/or that phenylalanine, tryptophan and/or tyrosineall have a generally similar shape. Therefore, based upon theseconsiderations, arginine, lysine and/or histidine; alanine, glycineand/or serine; and/or phenylalanine, tryptophan and/or tyrosine; aredefined herein as biologically functional equivalents.

To effect more quantitative changes, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and/or chargecharacteristics, these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (0.4); threonine (0.7); serine (0.8);tryptophan (0.9); tyrosine (1.3); proline (1.6); histidine (3.2);glutamate (3.5); glutamine (3.5); aspartate (3.5); asparagine (3.5);lysine (3.9); and/or arginine (4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte & Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index and/or score and/or stillretain a similar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and/or those within ±0.5 are even moreparticularly preferred.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biological functional equivalent protein and/orpeptide thereby created is intended for use in immunologicalembodiments, as in certain embodiments of the present invention. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and/or antigenicity, i.e., with a biological property ofthe protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (0.4);proline (−0.5±1); alanine (0.5); histidine (0.5); cysteine (1.0);methionine (1.3); valine (1.5); leucine (1.8); isoleucine (1.8);tyrosine (2.3); phenylalanine (2.5); tryptophan (3.4). In making changesbased upon similar hydrophilicity values, the substitution of aminoacids whose hydrophilicity values are within ±2 is preferred, thosewhich are within ±1 are particularly preferred, and/or those within ±0.5are even more particularly preferred.

B. Altered Amino Acids

The present invention, in many aspects, relies on the synthesis ofpeptides and polypeptides in cyto, via transcription and translation ofappropriate polynucleotides. These peptides and polypeptides willinclude the twenty “natural” amino acids, and post-translationalmodifications thereof. However, in vitro peptide synthesis permits theuse of modified and/or unusual amino acids. Table 1 provides exemplary,but not limiting, modified and/or unusual amino acids

C. Mimetics

In addition to the biological functional equivalents discussed above,the present inventors also contemplate that structurally similarcompounds may be formulated to mimic the key portions of peptide orpolypeptides of the present invention. Such compounds, which may betermed peptidomimetics, may be used in the same manner as the peptidesof the invention and, hence, also are functional equivalents.

Certain mimetics that mimic elements of protein secondary and tertiarystructure are described in Johnson et al. (1993). The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins exists chiefly to orient amino acid side chains insuch a way as to facilitate molecular interactions, such as those ofantibody and/or antigen. A peptide mimetic is thus designed to permitmolecular interactions similar to the natural molecule.

Some successful applications of the peptide mimetic concept have focusedon mimetics of β-turns within proteins, which are known to be highlyantigenic. Likely β turn structure within a polypeptide can be predictedby computer-based algorithms, as discussed herein. Once the componentamino acids of the turn are determined, mimetics can be constructed toachieve a similar spatial orientation of the essential elements of theamino acid side chains.

Other approaches have focused on the use of small,multidisulfide-containing proteins as attractive structural templatesfor producing biologically active conformations that mimic the bindingsites of large proteins. Vita et al. (1998). A structural motif thatappears to be evolutionarily conserved in certain toxins is small (30-40amino acids), stable, and high permissive for mutation. This motif iscomposed of a beta sheet and an alpha helix bridged in the interior coreby three disulfides.

Beta II turns have been mimicked successfully using cyclicL-pentapeptides and those with D-amino acids. Weisshoff et al. (1999).Also, Johannesson et al. (1999) report on bicyclic tripeptides withreverse turn inducing properties.

Methods for generating specific structures have been disclosed in theart. For example, alpha-helix mimetics are disclosed in U.S. Pat. Nos.5,446,128; 5,710,245; 5,840,833; and 5,859,184. Theses structures renderthe peptide or protein more thermally stable, also increase resistanceto proteolytic degradation. Six, seven, eleven, twelve, thirteen andfourteen membered ring structures are disclosed.

Methods for generating conformationally restricted beta turns and betabulges are described, for example, in U.S. Pat. Nos. 5,440,013;5,618,914; and 5,670,155. Beta-turns permit changed side substituentswithout having changes in corresponding backbone conformation, and haveappropriate termini for incorporation into peptides by standardsynthesis procedures. Other types of mimetic turns include reverse andgamma turns. Reverse turn mimetics are disclosed in U.S. Pat. Nos.5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S.Pat. Nos. 5,672,681 and 5,674,976.

VII. Immunological Compositions

In particular embodiments of the invention, immunological compositionsare employed. For the sake of brevity, the following section will referto any E. canis gp19 immunological compositions of the presentinvention, such as are described elsewhere herein as only exemplaryembodiments. For example, the compositions may include all or part of anE. canis gp19 polypeptide, such as one comprising part or all of SEQ IDNO:17 or SEQ ID NO:19, a gp19 polynucleotide, such as one comprisingpart or all of SEQ ID NO:16 or SEQ ID NO:18, a peptide, such as onecomprising SEQ ID NO:13, an antibody to a polypeptide or peptide of theinvention, or a mixture thereof, for example. Antibodies may be utilizedto bind an antigen, thereby rendering the molecule at least partiallyineffective for its activity, for example. In other embodiments,antibodies to the antigen are employed in diagnostic aspects of theinvention, such as for detecting the presence of the antigen from asample. Exemplary samples may be from an animal suspected of having E.canis or E. chaffeensis infection, from an animal susceptible to E.canis or E. chaffeensis infection, or from an animal that has an E.canis or E. chaffeensis infection. Exemplary samples may be obtainedfrom blood, serum, cerebrospinal fluid, urine, feces, cheek scrapings,nipple aspirate, and so forth.

Purified immunoreactive compositions or antigenic fragments of theimmunoreactive compositions can be used to generate new antibodies or totest existing antibodies (e.g., as positive controls in a diagnosticassay) by employing standard protocols known to those skilled in theart.

As is well known in the art, immunogenicity to a particular immunogencan be enhanced by the use of non-specific stimulators of the immuneresponse known as adjuvants. Exemplary and preferred adjuvants includecomplete BCG, Detox, (RIBI, Immunochem Research Inc.), ISCOMS andaluminum hydroxide adjuvant (Superphos, Biosector).

Included in this invention are polyclonal antisera generated by usingthe immunoreactive composition or a fragment of the immunoreactivecomposition as an immunogen in, e.g., rabbits. Standard protocols formonoclonal and polyclonal antibody production known to those skilled inthis art are employed. The monoclonal antibodies generated by thisprocedure can be screened for the ability to identify recombinantEhrlichia cDNA clones, and to distinguish them from known cDNA clones,for example.

The invention encompasses not only an intact monoclonal antibody, butalso an immunologically-active antibody fragment, e.g., a Fab or (Fab)2fragment; an engineered single chain scFv molecule; or a chimericmolecule, e.g., an antibody which contains the binding specificity ofone antibody, e.g., of murine origin, and the remaining portions ofanother antibody, e.g., of human origin.

In one embodiment, the antibody, or fragment thereof, may be linked to atoxin or to a detectable label, e.g. a radioactive label,non-radioactive isotopic label, fluorescent label, chemiluminescentlabel, paramagnetic label, enzyme label or colorimetric label. Examplesof suitable toxins include diphtheria toxin, Pseudomonas exotoxin A,ricin, and cholera toxin. Examples of suitable enzyme labels includemalate hydrogenase, staphylococcal nuclease, delta-5-steroid isomerase,alcohol dehydrogenase, alpha glycerol phosphate dehydrogenase, triosephosphate isomerase, peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholinesterase,etc. Examples of suitable radioisotopic labels include ³H, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C, etc.

Paramagnetic isotopes for purposes of in vivo diagnosis can also be usedaccording to the methods of this invention. There are numerous examplesof elements that are useful in magnetic resonance imaging. Fordiscussions on in vivo nuclear magnetic resonance imaging, see, forexample, Schaefer et al., (1989) JACC 14, 472-480; Shreve et al., (1986)Magn. Reson. Med. 3, 336-340; Wolf, G. L., (1984) Physiol. Chem. Phys.Med. NMR 16, 93-95; Wesby et al., (1984) Physiol. Chem. Phys. Med. NMR16, 145-155; Runge et al., (1984) Invest. Radiol. 19, 408-415. Examplesof suitable fluorescent labels include a fluorescein label, anisothiocyalate label, a rhodamine label, a phycoerythrin label, aphycocyanin label, an allophycocyanin label, an opthaldehyde label, afluorescamine label, etc. Examples of chemiluminiscent labels include aluminal label, an isoluminal label, an aromatic acridinium ester label,a luciferin label, a luciferase label, an aequorin label, etc.

Those of ordinary skill in the art will know of these and other suitablelabels, which may be employed in accordance with the present invention.The binding of these labels to antibodies or fragments thereof can beaccomplished using standard techniques commonly known to those ofordinary skill in the art. Typical techniques are described by Kennedyet al., (1976) Clin. Chim. Acta 70, 1-31; and Schurs et al., (1977)Clin. Chim. Acta 81, 1-40. Coupling techniques mentioned in the laterare the glutaraldehyde method, the periodate method, the dimaleimidemethod, the maleimidobenzyl-N-hydroxy-succinimde ester method. All ofthese methods are incorporated by reference herein.

D. Antibodies

In certain aspects of the invention, one or more antibodies may beproduced to the expressed gp36 or gp47. These antibodies may be used invarious diagnostic and/or therapeutic applications described herein.

As used herein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally,IgG and/or IgM are preferred because they are the most common antibodiesin the physiological situation and because they are most easily made ina laboratory setting.

The term “antibody” is used to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)2, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference).

“Mini-antibodies” or “minibodies” are also contemplated for use with thepresent invention. Minibodies are sFv polypeptide chains which includeoligomerization domains at their C-termini, separated from the sFv by ahinge region. Pack et al. (1992) Biochem 31:1579-1584. Theoligomerization domain comprises self-associating α-helices, e.g.,leucine zippers, that can be further stabilized by additional disulfidebonds. The oligomerization domain is designed to be compatible withvectorial folding across a membrane, a process thought to facilitate invivo folding of the polypeptide into a functional binding protein.Generally, minibodies are produced using recombinant methods well knownin the art. See, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumberet al. (1992) J Immunology 149B:120-126.

Antibody-like binding peptidomimetics are also contemplated in thepresent invention. Liu et al. Cell Mol Biol (Noisy-1e-grand). 2003March; 49(2):209-16 describe “antibody like binding peptidomimetics”(ABiPs), which are peptides that act as pared-down antibodies and havecertain advantages of longer serum half-life as well as less cumbersomesynthesis methods.

Monoclonal antibodies (MAbs) are recognized to have certain advantages,e.g., reproducibility and large-scale production, and their use isgenerally preferred. The invention thus provides monoclonal antibodiesof the human, murine, monkey, rat, hamster, rabbit and even chickenorigin. Due to the ease of preparation and ready availability ofreagents, murine monoclonal antibodies will often be preferred.

However, “humanized” antibodies are also contemplated, as are chimericantibodies from mouse, rat, or other species, bearing human constantand/or variable region domains, bispecific antibodies, recombinant andengineered antibodies and fragments thereof. As used herein, the term“humanized” immunoglobulin refers to an immunoglobulin comprising ahuman framework region and one or more CDR's from a non-human (usually amouse or rat) immunoglobulin. The non-human immunoglobulin providing theCDR's is called the “donor” and the human immunoglobulin providing theframework is called the “acceptor”. A “humanized antibody” is anantibody comprising a humanized light chain and a humanized heavy chainimmunoglobulin.

E. Exemplary Methods for Generating Monoclonal Antibodies

Exemplary methods for generating monoclonal antibodies (MAbs) generallybegin along the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal witha LEE or CEE composition in accordance with the present invention andcollecting antisera from that immunized animal.

A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. The choice ofanimal may be decided upon the ease of manipulation, costs or thedesired amount of sera, as would be known to one of skill in the art.Antibodies of the invention can also be produced transgenically throughthe generation of a mammal or plant that is transgenic for theimmunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies can beproduced in, and recovered from, the milk of goats, cows, or othermammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and5,741,957.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitableadjuvants include all acceptable immunostimulatory compounds, such ascytokines, chemokines, cofactors, toxins, plasmodia, syntheticcompositions or LEEs or CEEs encoding such adjuvants.

Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12,γ-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such asthur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A(MPL). RIBI, which contains three components extracted from bacteria,MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2%squalene/Tween 80 emulsion is also contemplated. MHC antigens may evenbe used. Exemplary, often preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

In addition to adjuvants, it may be desirable to coadminister biologicresponse modifiers (BRM), which have been shown to upregulate T cellimmunity or downregulate suppressor cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, Pa.);low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, N.J.),cytokines such as γ-interferon, IL-2, or IL-12 or genes encodingproteins involved in immune helper functions, such as B-7.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen including but not limited to subcutaneous, intramuscular,intradermal, intraepidermal, intravenous and intraperitoneal. Theproduction of polyclonal antibodies may be monitored by sampling bloodof the immunized animal at various points following immunization.

A second, booster dose (e.g., provided in an injection), may also begiven. The process of boosting and titering is repeated until a suitabletiter is achieved. When a desired level of immunogenicity is obtained,the immunized animal can be bled and the serum isolated and stored,and/or the animal can be used to generate MAbs.

For production of rabbit polyclonal antibodies, the animal can be bledthrough an ear vein or alternatively by cardiac puncture. The removedblood is allowed to coagulate and then centrifuged to separate serumcomponents from whole cells and blood clots. The serum may be used as isfor various applications or else the desired antibody fraction may bepurified by well-known methods, such as affinity chromatography usinganother antibody, a peptide bound to a solid matrix, or by using, e.g.,protein A or protein G chromatography.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified protein, polypeptide, peptide or domain, be it awild-type or mutant composition. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells.

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Rodents such as mice and rats are preferred animals, however, the use ofrabbit, sheep or frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986, pp. 60 61), but mice arepreferred, with the BALB/c mouse being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

The animals are injected with antigen, generally as described above. Theantigen may be mixed with adjuvant, such as Freund's complete orincomplete adjuvant. Booster administrations with the same antigen orDNA encoding the antigen would occur at approximately two-weekintervals.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible.

Often, a panel of animals will have been immunized and the spleen of ananimal with the highest antibody titer will be removed and the spleenlymphocytes obtained by homogenizing the spleen with a syringe.Typically, a spleen from an immunized mouse contains approximately 5×10⁷to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma producing fusion procedures preferably are non antibodyproducing, have high fusion efficiency, and enzyme deficiencies thatrender then incapable of growing in certain selective media whichsupport the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65 66, 1986; Campbell, pp. 75 83,1984). cites). For example, where the immunized animal is a mouse, onemay use P3 X63/Ag8, X63 Ag8.653, NS1/1.Ag 4 1, Sp210 Ag14, FO, NSO/U,MPC 11, MPC11×45 GTG 1.7 and S194/5XX0 Bul; for rats, one may useR210.RCY3, Y3 Ag 1.2.3, IR983F and 4B210; and U 266, GM1500 GRG2, LICRLON HMy2 and UC729 6 are all useful in connection with human cellfusions. See Yoo et al., J Immunol Methods. 2002 Mar. 1; 261(1-2):1-20,for a discussion of myeloma expression systems.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the 8azaguanine resistant mouse murine myeloma SP2/0 non producer cell line.

Methods for generating hybrids of antibody producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al., (1977). The use ofelectrically induced fusion methods is also appropriate (Goding pp. 7174, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. First, a sample of the hybridomacan be injected (often into the peritoneal cavity) into ahistocompatible animal of the type that was used to provide the somaticand myeloma cells for the original fusion (e.g., a syngeneic mouse).Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection. Theinjected animal develops tumors secreting the specific monoclonalantibody produced by the fused cell hybrid. The body fluids of theanimal, such as serum or ascites fluid, can then be tapped to provideMAbs in high concentration. Second, the individual cell lines could becultured in vitro, where the MAbs are naturally secreted into theculture medium from which they can be readily obtained in highconcentrations.

Further, expression of antibodies of the invention (or other moietiestherefrom) from production cell lines can be enhanced using a number ofknown techniques. For example, the glutamine synthetase and DHFR geneexpression systems are common approaches for enhancing expression undercertain conditions. High expressing cell clones can be identified usingconventional techniques, such as limited dilution cloning and Microdroptechnology. The GS system is discussed in whole or part in connectionwith European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 andEuropean Patent Application No. 89303964.4.

MAbs produced by either means may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asHPLC or affinity chromatography. Fragments of the monoclonal antibodiesof the invention can be obtained from the monoclonal antibodies soproduced by methods which include digestion with enzymes, such as pepsinor papain, and/or by cleavage of disulfide bonds by chemical reduction.Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer.

It is also contemplated that a molecular cloning approach may be used togenerate monoclonals. In one embodiment, combinatorial immunoglobulinphagemid libraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning using cells expressing the antigen and controlcells. The advantages of this approach over conventional hybridomatechniques are that approximately 10⁴ times as many antibodies can beproduced and screened in a single round, and that new specificities aregenerated by H and L chain combination which further increases thechance of finding appropriate antibodies. In another example, LEEs orCEEs can be used to produce antigens in vitro with a cell free system.These can be used as targets for scanning single chain antibodylibraries. This would enable many different antibodies to be identifiedvery quickly without the use of animals.

Another embodiment of the invention for producing antibodies accordingto the present invention is found in U.S. Pat. No. 6,091,001, whichdescribes methods to produce a cell expressing an antibody from agenomic sequence of the cell comprising a modified immunoglobulin locususing Cre-mediated site-specific recombination is disclosed. The methodinvolves first transfecting an antibody-producing cell with ahomology-targeting vector comprising a lox site and a targeting sequencehomologous to a first DNA sequence adjacent to the region of theimmunoglobulin loci of the genomic sequence which is to be converted toa modified region, so the first lox site is inserted into the genomicsequence via site-specific homologous recombination. Then the cell istransfected with a lox-targeting vector comprising a second lox sitesuitable for Cre-mediated recombination with the integrated lox site anda modifying sequence to convert the region of the immunoglobulin loci tothe modified region. This conversion is performed by interacting the loxsites with Cre in vivo, so that the modifying sequence inserts into thegenomic sequence via Cre-mediated site-specific recombination of the loxsites.

Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer, orby expression of full-length gene or of gene fragments in E. coli.

F. Antibody Conjugates

The present invention further provides antibodies against gp19 proteins,polypeptides and peptides, generally of the monoclonal type, that arelinked to at least one agent to form an antibody conjugate. In order toincrease the efficacy of antibody molecules as diagnostic or therapeuticagents, it is conventional to link or covalently bind or complex atleast one desired molecule or moiety. Such a molecule or moiety may be,but is not limited to, at least one effector or reporter molecule.Effector molecules comprise molecules having a desired activity, e.g.,cytotoxic activity. Non-limiting examples of effector molecules whichhave been attached to antibodies include toxins, anti-tumor agents,therapeutic enzymes, radio-labeled nucleotides, antiviral agents,chelating agents, cytokines, growth factors, and oligo- orpoly-nucleotides. By contrast, a reporter molecule is defined as anymoiety which may be detected using an assay. Non-limiting examples ofreporter molecules which have been conjugated to antibodies includeenzymes, radiolabels, haptens, fluorescent labels, phosphorescentmolecules, chemiluminescent molecules, chromophores, luminescentmolecules, photoaffinity molecules, colored particles or ligands, suchas biotin.

Any antibody of sufficient selectivity, specificity or affinity may beemployed as the basis for an antibody conjugate. Such properties may beevaluated using conventional immunological screening methodology knownto those of skill in the art. Sites for binding to biological activemolecules in the antibody molecule, in addition to the canonical antigenbinding sites, include sites that reside in the variable domain that canbind pathogens, B-cell superantigens, the T cell co-receptor CD4 and theHIV-1 envelope (Sasso et al., 1989; Shorki et al., 1991; Silvermann etal., 1995; Cleary et al., 1994; Lenert et al., 1990; Berberian et al.,1993; Kreier et al., 1991). In addition, the variable domain is involvedin antibody self-binding (Kang et al., 1988), and contains epitopes(idiotopes) recognized by anti-antibodies (Kohler et al., 1989).

Certain examples of antibody conjugates are those conjugates in whichthe antibody is linked to a detectable label. “Detectable labels” arecompounds and/or elements that can be detected due to their specificfunctional properties, and/or chemical characteristics, the use of whichallows the antibody to which they are attached to be detected, and/orfurther quantified if desired. Another such example is the formation ofa conjugate comprising an antibody linked to a cytotoxic or anticellular agent, and may be termed “immunotoxins”.

Antibody conjugates are generally preferred for use as diagnosticagents. Antibody diagnostics generally fall within two classes, thosefor use in in vitro diagnostics, such as in a variety of immunoassays,and/or those for use in vivo diagnostic protocols, generally known as“antibody directed imaging”.

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to antibodies (see, for e.g., U.S. Pat. Nos. 5,021,236;4,938,948; and 4,472,509, each incorporated herein by reference). Theimaging moieties used can be paramagnetic ions; radioactive isotopes;fluorochromes; NMR-detectable substances; X-ray imaging.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samariumytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention astatine²¹¹, ¹⁴carbon, ⁵¹chromium,³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen,iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus,rhenium186, rhenium188, ⁷⁵selenium, ³⁵sulphur, technicium99m and/oryttrium⁹⁰. ¹²⁵I is often being preferred for use in certain embodiments,and technicium^(99m) and/or indium¹¹¹ are also often preferred due totheir low energy and suitability for long range detection. Radioactivelylabeled monoclonal antibodies of the present invention may be producedaccording to well-known methods in the art. For instance, monoclonalantibodies can be iodinated by contact with sodium and/or potassiumiodide and a chemical oxidizing agent such as sodium hypochlorite, or anenzymatic oxidizing agent, such as lactoperoxidase. Monoclonalantibodies according to the invention may be labeled with technetium99mby ligand exchange process, for example, by reducing pertechnate withstannous solution, chelating the reduced technetium onto a Sephadexcolumn and applying the antibody to this column. Alternatively, directlabeling techniques may be used, e.g., by incubating pertechnate, areducing agent such as SNCl₂, a buffer solution such as sodium-potassiumphthalate solution, and the antibody. Intermediary functional groupswhich are often used to bind radioisotopes which exist as metallic ionsto antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetracetic acid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

Another type of antibody conjugates contemplated in the presentinvention are those intended primarily for use in vitro, where theantibody is linked to a secondary binding ligand and/or to an enzyme (anenzyme tag) that will generate a colored product upon contact with achromogenic substrate. Examples of suitable enzymes include urease,alkaline phosphatase, (horseradish) hydrogen peroxidase or glucoseoxidase. Preferred secondary binding ligands are biotin and/or avidinand streptavidin compounds. The use of such labels is well known tothose of skill in the art and are described, for example, in U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149and 4,366,241; each incorporated herein by reference.

Yet another known method of site-specific attachment of molecules toantibodies comprises the reaction of antibodies with hapten-basedaffinity labels. Essentially, hapten-based affinity labels react withamino acids in the antigen binding site, thereby destroying this siteand blocking specific antigen reaction. However, this may not beadvantageous since it results in loss of antigen binding by the antibodyconjugate.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter & Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used asantibody binding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody(U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein byreference). Monoclonal antibodies may also be reacted with an enzyme inthe presence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents or by reaction with an isothiocyanate. In U.S.Pat. No. 4,938,948, imaging of breast tumors is achieved usingmonoclonal antibodies and the detectable imaging moieties are bound tothe antibody using linkers such as methyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987). This approach has been reported to produce diagnosticallyand therapeutically promising antibodies which are currently in clinicalevaluation.

In another embodiment of the invention, the anti-gp36 antibodies arelinked to semiconductor nanocrystals such as those described in U.S.Pat. Nos. 6,048,616; 5,990,479; 5,690,807; 5,505,928; 5,262,357 (all ofwhich are incorporated herein in their entireties); as well as PCTPublication No. 99/26299 (published May 27, 1999). In particular,exemplary materials for use as semiconductor nanocrystals in thebiological and chemical assays of the present invention include, but arenot limited to those described above, including group II-VI, III-V andgroup IV semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS,MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP,GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlSb, PbS, PbSe, Ge and Si andternary and quaternary mixtures thereof. Methods for linkingsemiconductor nanocrystals to antibodies are described in U.S. Pat. Nos.6,630,307 and 6,274,323.

G. Immunodetection Methods

In still further embodiments, the present invention concernsimmunodetection methods for binding, purifying, removing, quantifyingand/or otherwise generally detecting biological components such asimmunoreactive polypeptides. The antibodies prepared in accordance withthe present invention may be employed to detect wild type and/or mutantproteins, polypeptides and/or peptides. The use of wild-type and/ormutant antibodies is contemplated. Some immunodetection methods includeenzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA),immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay,bioluminescent assay, and Western blot to mention a few. The steps ofvarious useful immunodetection methods have been described in thescientific literature, such as, e.g., Doolittle M H and Ben-Zeev O,1999; Gulbis B and Galand P, 1993; De Jager R et al., 1993; and Nakamuraet al., 1987, each incorporated herein by reference.

In general, the immunobinding methods include obtaining a samplesuspected of comprising protein, polypeptide and/or peptide, andcontacting the sample with a first anti-gp19 antibody in accordance withthe present invention, as the case may be, under conditions effective toallow the formation of immunocomplexes.

These methods include methods for purifying wild type and/or mutantproteins, polypeptides and/or peptides as may be employed in purifyingwild type and/or mutant proteins, polypeptides and/or peptides frompatients' samples and/or for purifying recombinantly expressed wild typeor mutant proteins, polypeptides and/or peptides. In these instances,the antibody removes the antigenic wild type and/or mutant protein,polypeptide and/or peptide component from a sample. The antibody willpreferably be linked to a solid support, such as in the form of a columnmatrix, and the sample suspected of containing the wild type or mutantprotein antigenic component will be applied to the immobilized antibody.The unwanted components will be washed from the column, leaving theantigen immunocomplexed to the immobilized antibody, which wild type ormutant protein antigen is then collected by removing the wild type ormutant protein and/or peptide from the column.

The immunobinding methods also include methods for detecting andquantifying the amount of a wild type or mutant protein reactivecomponent in a sample and the detection and quantification of any immunecomplexes formed during the binding process. Here, one would obtain asample suspected of comprising a wild type or mutant protein and/orpeptide or suspected of comprising an E. canis organism, and contact thesample with an antibody against wild type or mutant, and then detect andquantify the amount of immune complexes formed under the specificconditions.

In terms of antigen detection, the biological sample analyzed may be anysample that is suspected of containing a wild type or mutantprotein-specific antigen, such as a specimen, a homogenized tissueextract, a cell, separated and/or purified forms of any of the abovewild type or mutant protein-containing compositions, or even anybiological fluid that comes into contact with an E. canis organism uponinfection.

Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any proteinantigens present. After this time, the sample-antibody composition, suchas a tissue section, ELISA plate, dot blot or western blot, willgenerally be washed to remove any non-specifically bound antibodyspecies, allowing only those antibodies specifically bound within theprimary immune complexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

The antibody employed in the detection may itself be linked to adetectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined. Alternatively, the first antibody thatbecomes bound within the primary immune complexes may be detected bymeans of a second binding ligand that has binding affinity for theantibody. In these cases, the second binding ligand may be linked to adetectable label. The second binding ligand is itself often an antibody,which may thus be termed a “secondary” antibody. The primary immunecomplexes are contacted with the labeled, secondary binding ligand, orantibody, under effective conditions and for a period of time sufficientto allow the formation of secondary immune complexes. The secondaryimmune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the antibody is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

One method of immunodetection uses two different antibodies. A firststep biotinylated, monoclonal or polyclonal antibody is used to detectthe target antigen(s), and a second step antibody is then used to detectthe biotin attached to the complexed biotin. In that method the sampleto be tested is first incubated in a solution containing the first stepantibody. If the target antigen is present, some of the antibody bindsto the antigen to form a biotinylated antibody/antigen complex. Theantibody/antigen complex is then amplified by incubation in successivesolutions of streptavidin (or avidin), biotinylated DNA, and/orcomplementary biotinylated DNA, with each step adding additional biotinsites to the antibody/antigen complex. The amplification steps arerepeated until a suitable level of amplification is achieved, at whichpoint the sample is incubated in a solution containing the second stepantibody against biotin. This second step antibody is labeled, as forexample with an enzyme that can be used to detect the presence of theantibody/antigen complex by histoenzymology using a chromogen substrate.With suitable amplification, a conjugate can be produced which ismacroscopically visible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

The immunodetection methods of the present invention have evidentutility in the diagnosis and prognosis of conditions such as variousforms of hyperproliferative diseases, such as cancer, includingleukemia, for example. Here, a biological and/or clinical samplesuspected of containing a wild type or mutant protein, polypeptide,peptide and/or mutant is used. However, these embodiments also haveapplications to non-clinical samples, such as in the titering of antigenor antibody samples, for example in the selection of hybridomas.

H. ELISAs

As detailed above, immunoassays, in their most simple and/or directsense, are binding assays. Certain preferred immunoassays are thevarious types of enzyme linked immunosorbent assays (ELISAs) and/orradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections is also particularly useful. However, it will bereadily appreciated that detection is not limited to such techniques,and/or western blotting, dot blotting, FACS analyses, and/or the likemay also be used.

In one exemplary ELISA, the antibodies of the invention are immobilizedonto a selected surface exhibiting protein affinity, such as a well in apolystyrene microtiter plate. Then, a test composition suspected ofcontaining the wild type and/or mutant protein antigen, such as aclinical sample, is added to the wells. After binding and/or washing toremove non-specifically bound immune complexes, the bound wild typeand/or mutant protein antigen may be detected. Detection is generallyachieved by the addition of another antibody that is linked to adetectable label. This type of ELISA is a simple “sandwich ELISA”.Detection may also be achieved by the addition of a second antibody,followed by the addition of a third antibody that has binding affinityfor the second antibody, with the third antibody being linked to adetectable label.

In another exemplary ELISA, the samples suspected of containing the wildtype and/or mutant protein antigen are immobilized onto the well surfaceand/or then contacted with the antibodies of the invention. Afterbinding and/or washing to remove non-specifically bound immunecomplexes, the bound antibodies are detected. Where the initialantibodies are linked to a detectable label, the immune complexes may bedetected directly. Again, the immune complexes may be detected using asecond antibody that has binding affinity for the first antibody, withthe second antibody being linked to a detectable label.

Another ELISA in which the wild type and/or mutant proteins,polypeptides and/or peptides are immobilized, involves the use ofantibody competition in the detection. In this ELISA, labeled antibodiesagainst wild type or mutant protein are added to the wells, allowed tobind, and/or detected by means of their label. The amount of wild typeor mutant protein antigen in an unknown sample is then determined bymixing the sample with the labeled antibodies against wild type and/ormutant before and/or during incubation with coated wells. The presenceof wild type and/or mutant protein in the sample acts to reduce theamount of antibody against wild type or mutant protein available forbinding to the well and thus reduces the ultimate signal. This is alsoappropriate for detecting antibodies against wild type or mutant proteinin an unknown sample, where the unlabeled antibodies bind to theantigen-coated wells and also reduces the amount of antigen available tobind the labeled antibodies.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein or solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and/or antibodies with solutions such as BSA, bovine gammaglobulin (BGG) or phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. Preferably, this will be an enzymethat will generate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact orincubate the first and second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea, or bromocresolpurple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS),or H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generated, e.g., usinga visible spectra spectrophotometer.

I. Immunohistochemistry

The antibodies of the present invention may also be used in conjunctionwith both fresh-frozen and/or formalin-fixed, paraffin-embedded tissueblocks prepared for study by immunohistochemistry (IHC). The method ofpreparing tissue blocks from these particulate specimens has beensuccessfully used in previous IHC studies of various prognostic factors,and/or is well known to those of skill in the art (Brown et al., 1990;Abbondanzo et al., 1990; Allred et al., 1990).

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen“pulverized” tissue at room temperature in phosphate buffered saline(PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and/or pelleting again by centrifugation;snap-freezing in 70° C. isopentane; cutting the plastic capsule and/orremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and/or cutting 25-50 serial sections.

Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 hours fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and/or embedding the block in paraffin; and/or cutting upto 50 serial permanent sections.

J. Immunoelectron Microscopy

The antibodies of the present invention may also be used in conjunctionwith electron microscopy to identify intracellular tissue components.Briefly, an electron-dense label is conjugated directly or indirectly tothe antibody. Examples of electron-dense labels according to theinvention are ferritin and gold. The electron-dense label absorbselectrons and can be visualized by the electron microscope.

K. Immunodetection Kits

In still further embodiments, the present invention concernsimmunodetection kits for use with the immunodetection methods describedabove. As the antibodies are generally used to detect wild type and/ormutant proteins, polypeptides and/or peptides, the antibodies willpreferably be included in the kit. However, kits including both suchcomponents may be provided. The immunodetection kits will thus comprise,in suitable container means, a first antibody that binds to a wild typeand/or mutant protein, polypeptide and/or peptide, and/or optionally, animmunodetection reagent and/or further optionally, a wild type and/ormutant protein, polypeptide and/or peptide.

In preferred embodiments, monoclonal antibodies will be used. In certainembodiments, the first antibody that binds to the wild type and/ormutant protein, polypeptide and/or peptide may be pre-bound to a solidsupport, such as a column matrix and/or well of a microtitre plate.

The immunodetection reagents of the kit may take any one of a variety offorms, including those detectable labels that are associated with and/orlinked to the given antibody. Detectable labels that are associated withand/or attached to a secondary binding ligand are also contemplated.Exemplary secondary ligands are those secondary antibodies that havebinding affinity for the first antibody.

Further suitable immunodetection reagents for use in the present kitsinclude the two-component reagent that comprises a secondary antibodythat has binding affinity for the first antibody, along with a thirdantibody that has binding affinity for the second antibody, the thirdantibody being linked to a detectable label. As noted above, a number ofexemplary labels are known in the art and/or all such labels may beemployed in connection with the present invention.

The kits may further comprise a suitably aliquoted composition of thewild type and/or mutant protein, polypeptide and/or polypeptide, whetherlabeled and/or unlabeled, as may be used to prepare a standard curve fora detection assay. The kits may contain antibody-label conjugates eitherin fully conjugated form, in the form of intermediates, and/or asseparate moieties to be conjugated by the user of the kit. Thecomponents of the kits may be packaged either in aqueous media and/or inlyophilized form.

The container means of the kits will be suitable housed and willgenerally include at least one vial, test tube, flask, bottle, syringeand/or other container means, into which the antibody may be placed,and/or preferably, suitably aliquoted. Where wild type and/or mutantgp19 protein, polypeptide and/or peptide, and/or a second and/or thirdbinding ligand and/or additional component is provided, the kit willalso generally contain a second, third and/or other additional containerinto which this ligand and/or component may be placed. The kits of thepresent invention will also typically include a means for containing theantibody, antigen, and/or any other reagent containers in closeconfinement for commercial sale. Such containers may include injectionand/or blow-molded plastic containers into which the desired vials areretained.

VIII. Pharmaceutical Preparations

It is also contemplated that pharmaceutical compositions may be preparedusing the novel compositions of the present invention. In such a case,the pharmaceutical composition comprises the novel active composition ofthe present invention and a pharmaceutically acceptable carrier. Aperson having ordinary skill in this art would readily be able todetermine, without undue experimentation, the appropriate dosages androutes of administration of the active component of the presentinvention.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a subject. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

In general, a pharmaceutical composition of the present invention maycomprise an E. canis gp19 polypeptide, polynucleotide, or antibodyand/or mixtures thereof.

A protein may be formulated into a composition in a neutral or saltform. Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids such as acetic, oxalic, tartaric, mandelic,and the like. Salts formed with the free carboxyl groups can also bederived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, histidine, procaine and the like.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media, which can be employed, will be knownto those of skill in the art in light of present disclosure. Forexample, one dosage could be dissolved in 1 mL of isotonic NaCl solutionand either added to 1000 mL of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more agents that target a polypeptide or thesecretion thereof or additional agent dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical,”“pharmaceutically acceptable,” or “pharmacologically acceptable” refersto molecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas, for example, a human, as appropriate. The preparation of apharmaceutical composition that contains at least one agent that targetsthe polypeptide or the secretion thereof and/or additional activeingredient will be known to those of skill in the art in light of thepresent disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. Moreover, for animal (e.g., human) administration, it will beunderstood that preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The invention may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. The present invention can be administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, topically, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, inhalation (e.g. aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in cremes, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art (see,for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack PrintingCompany, 1990, incorporated herein by reference).

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The invention may be formulated into a composition in a free base,neutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays,aerosols or inhalants in the present invention. Such compositions aregenerally designed to be compatible with the target tissue type. In anon-limiting example, nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,in preferred embodiments the aqueous nasal solutions usually areisotonic or slightly buffered to maintain a pH of about 5.5 to about6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments the composition is prepared for administration bysuch routes as oral ingestion. In these embodiments, the solidcomposition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions may be incorporated directly with the food of the diet.Preferred carriers for oral administration comprise inert diluents,assimilable edible carriers or combinations thereof. In other aspects ofthe invention, the oral composition may be prepared as a syrup orelixir. A syrup or elixir, and may comprise, for example, at least oneactive agent, a sweetening agent, a preservative, a flavoring agent, adye, a preservative, or combinations thereof.

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Additional formulations that are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle that contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

IX. Exemplary Kits of the Invention

In particular embodiments of the invention, there is a kit housed in asuitable container. The kit may be suitable for diagnosis, treatment,and/or protection for an individual from Ehrlichia, such as Ehrlichiacanis. In particular embodiments, the kit comprises in a suitablecontainer an agent that targets an E. canis gp19 antigen. The agent maybe an antibody, a small molecule, a polynucleotide, a polypeptide, apeptide, or a mixture thereof. The agent may be provided in the kit in asuitable form, such as sterile, lyophilized, or both, for example. Inparticular embodiments, the kit comprises an antibody against one ormore of SEQ ID NO:13, SEQ ID NO:17 or SEQ ID NO:19 (for E. canis);and/or related proteins thereof. Other E. canis gp19-relatedimmunogenic-related compositions (including polypeptides, peptides, orantibodies) not specifically presented herein may also be included.

The kit may further comprise one or more apparatuses for delivery of acomposition to an individual in need thereof. The apparatuses mayinclude a syringe, eye dropper, needle, biopsy tool, scoopula, catheter,and so forth, for example.

In embodiments wherein the kit is employed for a diagnostic purpose, thekit may further provide one or more detection compositions and/orapparatuses for identifying an E. canis gp19 antigen. Such an embodimentmay employ a detectable label, such as for an antibody, for example, andthe label may be fluorescent, radioactive, chemiluminescent, orcolorimetric, for example.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Exemplary Materials and Methods

Culture and purification of Ehrlichiae. E. canis (Jake, DJ, Demon,Louisiana, Florida, and Sao Paulo strains) and were propogated aspreviously described (McBride et al., 2001). Ehrlichiae were purified bysize exclusion chromatography over Sephacryl S-1000 (AmershamBiosciences, Piscataway, N.J.) as previously described (Rikihisa et al.,1992). The fractions containing bacteria were frozen and utilized asantigen and DNA sources.

Construction and screening of the E. canis genomic library. An E. canisJake strain genomic library was constructed using a HpaII restrictiondigest and screened as previously described (McBride et al., 2001).

DNA sequencing. Library inserts, plasmids, and PCR products weresequenced with an ABI Prism 377XL DNA Sequencer (Perkin-Elmer AppliedBiosystems, Foster City, Calif.) at the University of Texas MedicalBranch Protein Chemistry Core Laboratory.

Glycoprotein analysis. Nucleic acid and amino acid alignments wereperformed with MegAlign (Lasergene v5.08, DNAstar, Madison, Wis.). TheE. canis gp19 and E. chaffeensis VLPT protein sequences were evaluatedfor potential O-linked glycosylation and phosphorylation with thecomputational algorithms YinOYang 1.2 and NetOGlyc v3.1 (Julenius etal., 2005). Tandem Repeat Finder (Benson, 1999) was used to analyze thetandem repeats of the genes encoding E. canis gp19. Potential signalsequences were identified with the computational algorithm SignalPtrained on gram-negative bacteria (Nielsen et al., 1997).

PCR amplification of the E. canis gp19 gene fragments. Oligonucleotideprimers for the amplification of the E. canis gp19 gene fragments (gp19;gp19N-terminal, gp19N1, gp19N2, gp19N1-C, gp19C-terminal) were designedmanually or by using Primer Select (Lasergene v5.08, DNAstar, MadisonWis.) (Table 2). E. canis gp19 fragments were amplified using the PCRMaster mix (F. Hoffmann-La Roche Ltd, Basel, Switzerland) and E. canis(Jake strain) genomic DNA as template.

TABLE 2Exemplary oligonucleotide primers for amplification of E. canis gp19 geneRe- combinant Forward Reverse Amplicon Protein Primer Sequence PrimerSequence Size gp19 P16N-F 5′-CACGTTCAAAATCATGTTGA-3′ P16C-R5′-CGCACAATCACAACAGTTGT-3 405 bp (SEQ ID NO: 1) (SEQ ID NO: 7) gp19NP16N-F 5′-CACGTTCAAAATCATGTTGA-3′ P16N-R 5′-GCATACTGGTCTTTCCT-3′ 222 bp(SEQ ID NO: 2) (SEQ ID NO: 8) gp19N1 P16N-F 5′-CACGTTCAAAATCATGTTGA-3′p19 132-R 5′-AGATACTTCTTGTAACTCCATT-3′ 126 bp (SEQ ID NO: 3)(SEQ ID NO: 9) gp19N1-C Small FOR 5′-CATTTTACTGGTCCTACT-3′ p19 132-R5′-AGATACTTCTTGTAACTCCATT-3′  72 bp (SEQ ID NO: 4) (SEQ ID NO: 10)gp19N2 p19 133-F 5′-TCTATTGATAGTGTAGGATGC-3′ P16N-R5′-GCATACTGGTCTTTCCT-3′  96 bp (SEQ ID NO: 5) (SEQ ID NO: 11) gp19CP16C-F 5′-GCAGGTTTAGAGAGCTT-3′ P16C-R 5′-CGCACAATCACAACAGTTGT-3 180 bp(SEQ ID NO: 6) (SEQ ID NO: 12)

Cloning and expression of recombinant E. canis gp19. The amplified PCRproducts were cloned directly into the pBAD/TOPO Thio Fusion® or pCRT7/NT TOPO expression vector (Invitrogen, Carlsbad, Calif.). E. coli(TOP10, Invitrogen) were transformed with the plasmid containing the E.canis gp19 gene fragments, and positive transformants were screened byPCR for the presence of the insert and its orientation and weresequenced to confirm the reading frame of the genes. Recombinant proteinexpression was induced with 0.2% arabinose (pBAD/TOPO Thio Fusion), orIPTG (pCR T7/NT) using the Overnight Express Autoinduction System 1(Novagen, Madison, Wis.). Bacteria were pelleted (5,000×g for 20 min),resuspended in PBS, and recombinant proteins were purified under nativeconditions as previously described (Doyle et al., 2005).

Gel electrophoresis and Western immunoblotting. Purified E. canis or E.chaffeensis whole cell lysates or recombinant proteins were separated bySDS-PAGE, transferred to nitrocellulose, and Western blots performed aspreviously described (McBride et al., 2003), except primary antibodieswere diluted (1:500). Anti-E. canis or E. chaffeensis dog sera werederived from experimentally infected dogs (#2995 and #2551,respectively).

Carbohydrate detection and glycosyl composition. Glycan detection on therecombinant protein gp19 was performed with a DIG glycan detection kit(Roche, Indianapolis, Ind.) as previously described (McBride et al.,2000). The glycosyl composition was determined by alditol acetateanalysis at the University of Georgia Complex Carbohydrate ResearchCenter. The glycoprotein was hydrolyzed using 2 M trifluoroacetic acid(TFA; 2 h in sealed tube at 121° C.), reduced with NaBD4, and acetylatedusing acetic anhydride/TFA. The resulting alditol acetate was analyzedon a Hewlett Packard 5890 gas chromatograph interfaced to a 5970 massselective detector (electron impact ionization mode), and separation wasperformed on a 30 m Supelco 2330 bonded phase fused silica capillarycolumn.

Mouse immunization. Five BALB/c mice (Jackson Laboratories, Bar Harbor,Me.) were immunized with the recombinant E. canis gp19 (Thio fusion;amino acids 4 to 137). Recombinant protein (100 μg) in 0.1 mL was mixedwith an equal volume of Freund's complete adjuvant (Sigma, St. Louis,Mo.) for the first intraperitoneal injection and with Freund'sincomplete adjuvant for the subsequent injections. The mice were giveninjections twice at two week intervals.

E. canis gp19 synthetic peptide antibody epitope. A 24 amino acidpeptide (N1-C; HFTGPTSFEVNLSEEEKMELQEVSS; SEQ ID NO:13) corresponding tothe E. canis gp19 epitope-containing region was synthesized byBio-Synthesis, Inc. (Lewisville, Tx).

Enzyme-linked immunosorbent assay (ELISA). ELISA plates (Nunc-Immuno™Plates with MaxiSorp™ Surface, NUNC, Roskilde, Denmark) were coated withrecombinant protein or peptide (1.25 μg/well, 100 μL) in phosphatebuffered saline (PBS). Antigen was adsorbed to the ELISA platesovernight at 4° C. with gentle agitation and subsequently washed threetimes with 200 μL Tris buffered saline with Tween 20 (0.2%) (TBST),blocked with 3% BSA in TBST for 1 hr at room temperature with agitationand washed again. Convalescent anti-E. canis canine serum (1:4000)diluted in 3% BSA TBST was added to each well (100 μL) and incubated atroom temperature for 1 h with gentle agitation. The plates were washedfour times, and an alkaline phosphatase-labeled goat anti-dog IgG (H+L)secondary antibody (1:2500) (Kirkegaard & Perry Laboratories) in 3% BSATBST was added and incubated for 1 hr. The plates were washed fourtimes, and substrate (100 μL) (BluePhos, Kirkegaard & PerryLaboratories) was added to each well. The plates were incubated for 30min in the dark with agitation, and color development was read on amicroplate reader (Versamax, Molecular Devices, Sunnyvale, Calif.) atA650 and data analyzed by SoftmaxPro v4.0 (Molecular Devices). Opticaldensity readings of the bar graph represent the mean of two wells withthe O.D. of the buffer-only wells subtracted. Periodate treatment of therecombinant gp19 was carried out for 20 min in 100 mM sodium acetatebuffer with 100 mM sodium metaperiodate. Sham-treated control proteinwas incubated in the same buffers in the absence of periodate. The ELISAwas carried out as described above for E. canis antigens, with theexception that the plate was blocked with milk diluent/blocking solution(Kirkegaard & Perry Laboratories, Gaithersburg, Md.).

Immunoelectron microscopy. Immunogold electron microscopy was performedas previously described (Doyle et al., 2005) except primary anti-E.canis gp19 antibody was diluted 1:10,000. Uninfected DH82 cells werereacted with anti-E. canis gp19 as a negative control.

Fluorescent confocal microscopy. Antigen slides were prepared from DH82cells infected with E. canis (Jake strain) as described previously(McBride et al., 2001). Monospecific rabbit serum against therecombinant E. canis disulfide bond formation protein (DsbA) (McBride etal., 2002) diluted 1:100 was added to each well (15 μL) and allowed toincubate for 30 min. Slides were washed, and mouse anti-gp19 (1:100dilution) was added and incubated for 30 min. Alexa Fluor® 488 goatanti-rabbit IgG (H & L) secondary antibody (Molecular Probes, Eugene,Or.) diluted 1:100 was added and incubated for 30 min, followed bywashing and subsequent addition and incubation of rhodamine-labeled goatanti-mouse IgG (H & L) secondary antibody (Kirkegaard & PerryLaboratories). Mounting medium (ProLong Gold, Molecular Probes) wasadded and the slides were viewed with an Olympus FV-1000 laser confocalmicroscope and Fluoview™ software.

PCR amplification of E. canis gp19 from geographically dispersedisolates. DNA isolated from North American (Jake, Demon, DJ, Louisianaand Florida) isolates, South American (Brazil) isolate (Sao Paulo;kindly provided by Marcelo Labruna) and infected dog from Mexico(Yucatan; kindly provided by Carlos Perez) were used as template toamplify the entire gp19 gene using flanking primers (Forward-Ecanis p19FOR, 5′-AAAATTAGTGTTGTGGTTATG-3′ (SEQ ID NO:14) and reverse-Ecanis p19REV, 5′-TTTTACGCTTGCTGAAT-3′; SEQ ID NO:15). The amplicons were clonedinto a TA cloning vector (pCR 2.1, Invitrogen) and plasmids transformedinto E. coli (TOP10). Plasmids containing gp19 were purified withplasmid purification kit (Roche) and sequenced.

Example 2 Molecular Identification of the E. canis gp19 MajorImmunoreactive Protein

Screening of an E. canis genomic expression library identified a clonethat reacted strongly with antibody and contained a ˜3-kb insert. Thisclone was partially sequenced (−900 bp) to reveal an incomplete ORF,which was aligned with the available E. canis genome sequence to fullyidentify the genes present within the 3 kb clone. The clone contained acomplete 1086-bp gene encoding a riboflavin biosynthesis protein (RibD),and a downstream 414-bp ORF encoding a protein of 137 amino acids with apredicted mass of 15.8 kDa with unknown function. The protein had a 47amino acid C-terminal region with >53% identity and ˜60% overallhomology to E. chaffeensis VLPT, a known immunoreactive protein;therefore this gene was considered for further investigation. The E.canis protein had substantial C-terminal region homology (60%) with E.chaffeensis VLPT, but it lacked the characteristic tandem repeats. TheE. canis protein did have several predicted O-glycan attachment sitesand one amino acid (serine 44) that was a predicted Yin-Yang site(glycosylation/phosphorylation). Further analysis of the gene positionin the chromosome revealed the same adjacent genes for the 414-bp E.canis gene and that of E. chaffeensis vlpt (FIG. 1).

Protein characteristics. Cysteine (14; 10.2%), serine and threonine (13;9.5% combined), glutamate (13; 9.5%) and tyrosine (13; 9.5%) were themost frequently occurring amino acids in the E. canis gp19, accountingfor more 38% of the entire amino acid content. Cysteine residues werenot present in the first 50 amino acids, but the carboxyl-terminalregion of the protein (last 28 amino acids) was dominated by cysteineand tyrosine (55%). Serine, threonine (7 each; 27% and glutamateresidues (6; 23%) were the most frequently occurring amino acids in asmall central region (STE-rich patch; 26 amino acids) and accounted for50% of the amino acid content.

Conservation of E. canis gp19. E. canis gp19 was examined ingeographically dispersed North American (Jake, DJ, Demon, Louisiana, andFlorida) and South American (Brazil; Sao Paulo) isolates and wascompletely conserved. The gp19 sequence amplified from an E.canis-infected dog from Mexico (Yucatan) had a single nucleotidesubstitution (position 71) that resulted in a single amino acid changefrom glycine to aspartate.

Molecular mass and immunoreactivity. The mass of the gp19 fusionrecombinant protein was ˜35 kDa, and was larger (˜3 kDa) than thepredicted (32 kDa) mass which included the fusion tags (13 kDa), but wasconsistent with the ˜3 kDa larger than predicted (16 kDa) mass of thenative gp19 (19-kDa) (FIG. 2A). Similarly, smaller fragments of the gp19(N-terminal, N1 and N1c) expressed as recombinant fusion proteins hadmolecular masses larger (˜6 kDa) (see FIG. 5 for orientation) thanpredicted by their amino acid sequences. The recombinant gp19 reactedstrongly with serum from a dog (#2995) experimentally infected with E.canis (FIG. 2B).

Carbohydrate detection. Carbohydrate was detected on the recombinantgp19 (N-terminal, see FIG. 4 for orientation), which contained theSTE-patch (FIG. 3). Furthermore, glycosyl composition analysis of theN-terminal fragment by the University of Georgia Complex CarbohydrateResearch Center using alditol acetate analysis revealed the presence ofglucose and xylose.

Identification of native gp19 and species specificity. Anti-recombinantgp19 antisera reacted strongly with a 19 kDa protein in E. canis wholecell lysates, and this protein was similarly recognized by anti-E. canisdog serum (FIG. 4A). The anti-recombinant gp19 sera also reacted weaklywith another well characterized E. canis glycoprotein, gp36, suggestinga minor cross reactivity between these two proteins. Theanti-recombinant gp19 sera did not recognize antigens in E. chaffeensiswhole cell lysates (FIG. 4B).

Single major epitope. Epitope determinants of other glycoproteins havebeen determined including the E. chaffeensis gp47 and E. canis gp36(Doyle et al., 2006). The E. canis gp19 is strongly recognized byantibody of infected dogs, and it elicits an early antibody response(McBride et al., 2003). In order to identify the epitope-containingregion, E. canis gp19 gene fragments (N-terminal, C-terminal, N1, N2,N1C) (FIG. 5) were amplified with primers (Table 2) to createoverlapping recombinant fusion proteins. The expressed gp19 fragments(N1, N2, N-terminal and C-terminal) exhibited larger (2- to 6-kDa) thanpredicted masses by SDS-PAGE (FIG. 4A). Antibody reacted strongly withthe N-terminal recombinant fragment, but did not react with theC-terminal fragment indicating that an epitope was located in theN-terminal region of the protein (FIG. 5B). Further localization of theepitope containing region was determined with fragments N1 and N2.Antibody strong reacted with the N1 (42 amino acids), and N2 was weaklyrecognized (FIG. 5B). A region within N1 that had a high Ser/Thr/Glucontent (N1C; 24 amino acids) consistent with other epitopes identifiedin other ehrlichial proteins reacted strongly with antibody consistentwith that of the larger N1 fragment, demonstrating that a single majorepitope was located in the 24 amino acid region of N1C.

Carbohydrate as an epitope determinant. It was previously shown thatcarbohydrate is an important epitope determinant on major immunoreactiveglycoproteins (Doyle et al., 2006). Carbohydrate was detected on theN-terminal region of the gp19, and the epitope localized to the STE-richpatch. Glycan attachment sites were also predicted within the STE-richpatch. To determine the role of carbohydrate determinants in antibodyrecognition, the immunoreactivity of recombinant N1C was compared withthat of synthetic peptide. By ELISA, the synthetic peptide wassubstantially less immunoreactive with anti-E. canis dog serum (#2995)than the recombinant version (FIG. 6). Similarly, N1C treated withperiodate to alter glycan structure was less immunoreactive than shamtreated N1C protein (FIG. 6).

Cellular and extracellular localization of gp19. Several characterizedehrlichial glycoproteins are differentially expressed on dense-coredEhrlichiae (gp120, gp36 and gp47). However, by immunoelectron microscopythe E. canis gp19 was observed within the cytoplasm of both reticulateand dense cored Ehrlichiae, but was also detected extracellularly on themorula fibrillar matrix and associated with the morula membrane (FIG.7). These results were consistent with observations using confocalimmunoflourescent microscopy using anti-gp19 (FIG. 8A) and anti-Dsbantibody (present on Ehrlichiae, but not extracellularly) (FIG. 8B),showing both Dsb and gp19 colocalizing on Ehrlichiae, and the borderstaining of the morula membrane by anti-gp19 only (merged) (FIG. 8C).

Nucleotide sequence accession numbers. The Ehrlichia canis gp19 genesequences from E. canis gp19 (Jake, DJ, Demon, Louisiana, Florida, SaoPaulo and Mexico) isolate were deposited into GenBank® and assigned thefollowing respective accession numbers: DQ858221, DQ858222, DQ858223,DQ858224, DQ858225, DQ860145, and DQ858226. All of these Accessionnumbers are represented in the polynucleotide sequence of SEQ ID NO:16and the polypeptide sequence of SEQ ID NO:17 except the GenBank®accession number DQ858226, which is represented in the polynucleotidesequence of SEQ ID NO:18 and the polypeptide sequence of SEQ ID NO:19.

Example 3 Significance of the Present Invention

The kinetics of antibody responses to major immunoreactive antigens ofE. canis during experimental infection has been well established in aprevious study (McBride et al., 2003). Two E. canis antigens (37- and19-kDa) were consistently recognized early in the acute immune response.In a more a recent study, the identification and molecularcharacterization of the 37-kDa protein (gp36), which is a differentiallyexpressed glycoprotein on dense-cored Ehrlichiae and is secreted, wasdescribed (Doyle et al., 2006). As more major immunoreactive proteinshave been molecularly characterized in E. canis and E. chaffeensis, ithas become apparent that many exhibit high serine/threonine content,contain tandem repeats and are glycosylated (Doyle et al., 2006; McBrideet al., 2000; Yu et al., 1997; Yu et al., 2000).

Although others have reported that orthologs of E. chaffeensis vlpt werenot identified in related genomes (E. canis and E. ruminantium) (Hotoppet al., 2006), we provide evidence herein that the 19-kDa proteinidentified in this study is the ortholog of the previous described VLPTprotein in E. chaffeensis (Sumner et al., 1999). The E. chaffeensis VLPTis immunoreactive, and has non-identical serine-rich tandem repeats.Although carbohydrate has not been reported on the E. chaffeensis VLPT,the protein also exhibits a mass double that predicted by its amino acidcontent, similar to other described ehrlichial glycoproteins (Sumner etal., 1999). Interestingly, the vlpt ortholog that we identified in E.canis in this study lacks the tandem repeats found in E. chaffeensisvlpt, but has a Ser/Thr/Glu-rich patch that is similar is size andcomposition to that of a single VLPT repeat unit. In addition, thesegenes share the same chromosomal location and have substantial aminoacid homology (˜60%) in the carboxyl-terminal region.

Another major immunoreactive protein (MAP2) related to Anaplasmamarginale MSP5 has been identified and molecularly characterized in E.canis, E. chaffeensis and E. ruminantium with a molecular mass (˜21 kDa)similar to the gp19 identified in this study (Alleman et al., 2000;Alleman et al., 2001; Mahan et al., 1994). However, there is no aminoacid homology between MAP2 and gp19, and thus, these proteins aremolecularly and immunologically distinct. Unlike the gp19, the MAP2appears to have a mass consistent with that predicted by its amino acidsequence and does not have any serine-rich domains. There is substantialhomology among MAP2 orthologs in Ehrlichia spp., and cross reactionsamong heterologous MAP2 proteins have been reported (Knowles et al.,2003; Mahan et al., 1994). In contrast, antibodies generated to the E.canis gp19 were not cross reactive with E. chaffeensis VLPT, andtherefore these proteins appear to be species-specific orthologs. Othernotable differences between MAP2 and gp19 include a major serine-richlinear epitope of gp19 that is strongly recognized by antibodies byWestern immunoblot, while antibodies to the MAP2 of E. canis and E.chaffeensis appear to directed primarily at a conformational epitope(Alleman et al., 2000; Alleman et al., 2001; Knowles et al., 2003). In aprevious study it was suggested that the 19-kDa major immunoreactiveprotein that was identified may be MAP2 (McBride et al., 2003); however,data presented in this invention indicates that this protein is notMAP2, but rather gp19. Interestingly, only one major immunoreactiveprotein in the range of 15- to 25-kDa was identified in the previousstudy (McBride et al., 2003). The fact that antibodies to MAP2 wereunable to be detected is likely related to the fact that conformationalepitopes are dominant on both E. canis and E. chaffeensis MAP2 (Allemanet al., 2000; Alleman et al., 2001).

Consistent with numerous other major immunoreactive proteins that havebeen characterized, carbohydrate was present on the N-terminal region ofthe E. canis 19 kDa protein, and glucose and xylose were detected onthis fragment. The presence of glucose and galactose as sugars attachedto the E. chaffeensis gp120 and E. canis gp140 has been reported.Although the E. canis gp19 does not have serine-rich tandem repeats thatappear to be locations of glycan attachment, it did contain a STE-richpatch within the N-terminal region, similar to the amino acidcomposition of tandem repeats found in other ehrlichial glycoproteins.Therefore, it is likely that O-linked glycans are attached to aminoacids (serine/threonine) in this STE-rich patch. Furthermore, by usingthe prediction server YinOYang, serine residues within this region wereidentified as potential glycosylation/phosphorylation sites. Since thisprediction server is trained on eukaryotic glycoproteins, identificationof specific residues that are glycosylated may not be reliable; however,it is worth noting that there is a consistent positive correlationbetween our experimental data the prediction generated by thiseukaryote-based prediction algorithm.

The amino acid composition of the E. canis gp19 consisted predominatelyof five amino acids, cysteine, glutamate, tyrosine, serine andthreonine. Interestingly, these amino acids were concentrated in twospecific domains, the epitope-containing region and carboxyl-terminalregion. The high Ser/Thr/Glu content of the epitope containing regionhas been reported in other ehrlichial glycoproteins where epitopes havebeen mapped (Doyle et al., 2006), and high serine and threonine contenthas been found in other ehrlichial glycoproteins, particularly in tandemrepeat regions (Doyle et al., 2006; Yu et al., 1997; Yu et al., 2000).The E. chaffeensis VLPT also contains tandems repeats with similar aminoacid content. This similarity indicates that this region within the E.canis gp19 corresponds to the tandem repeat in E. chaffeensis VLPT. Theaddition and deletion of tandem repeats is considered a major source ofchange and instability in ehrlichial genomes (Frutos et al., 2006). Thefact that the E. canis gp19 lacks tandem repeats, while E. chaffeensisVLPT has variable numbers is indicative of these genes being affected bythis process.

Another novel feature of the gp19 is a carboxyl-terminal tail dominatedby tyrosine and cysteine (55%). This carboxyl-terminal tail was alsopresent on the E. chaffeensis VLPT downstream of the repeat region,indicating that it is an important conserved domain in these proteins.Overall, cysteine was present more than any other amino acid, andbecause of this, the gp19 is a member of a small group of proteins(n=36) with high cysteine content (Mavromatis et al., 2006). Cysteine isessential for intra- and inter-molecular disulfide bond formation, andits high content in the gp19 indicates that this protein has thepotential to be linked with other cysteine containing proteins bydisulfide bonds or that they are important for intramolecular bondingnecessary for maintaining gp19 structure.

Tyrosine and serine are commonly phosphorylated. The high proportion oftyrosine residues in the carboxyl-terminal region of the gp19 suggests ahigh potential for this domain of the protein to be phosphorylated. Thiscondition also raises the possibility that the gp19 is involved inprotein signaling. The presence of phosphoproteins has been reported inE. chaffeensis (Singu et al., 2005), and more Ser/Thr/Tyr kinases andphosphoproteins are being identified in bacteria (Hinc et al., 2006;Levine et al., 2006; Madec et al., 2002; Obuchowski et al., 2000).Nevertheless, tyrosine residues in this C-terminal region were notidentified as sites of phosphorylation by NetPhos, which is trained oneukaryote proteins. Therefore, further studies are performed tocharacterize the phosphorylation status of tyrosine residues how thisrelates to protein function, in specific embodiments of the invention.

A single major epitope was identified in the E. canis gp19 in theSTE-rich domain. This epitope elicits an early antibody response in dogsexperimentally infected with E. canis (McBride et al., 2003). Otherepitopes that we have characterized within ehrlichial glycoproteins weremapped to serine-rich tandem repeats. Hence, finding a major epitopewithin the STE-region of the gp19 is consistent with previous studies,and demonstrates the importance of serine-rich regions and attachedcarbohydrate as immunodeterminants for Ehrlichia spp. Carbohydrate wasdetected on N-terminal region of the gp19 containing the STE-region. Therecombinant gp19 epitope was more immunoreactive than the correspondingsynthetic peptide, indicating that a post-translational modification waspresent on this epitope. In addition, treatment of the recombinantepitope-containing peptide with periodate reduced its immunoreactivity,further supporting a role for carbohydrate as an immunodeterminant.These findings are consistent with the previous demonstration ofcarbohydrate as an immunodeterminant on the epitopes that were mapped inthe serine-rich tandem repeat regions of E. chaffeensis gp47 and E.canis gp36 (Doyle et al., 2006). Notably, this epitope appears to bespecies-specific, and the anti-gp19 antibody did not crossreact with E.chaffeensis antigens, similar to other species-specific majorimmunoreactive antigens that have been identified including the gp36(Doyle et al., 2006; Yu et al., 1997; Yu et al., 2000). Therefore, useof sensitive species-specific immunodiagnostics utilizing the E. canisgp19 alone, or in combination with other antigens such as the gp36, arespecific embodiments of the invention.

The E. canis gp19 was found on both reticulate and dense-cored cells andappeared to be localized predominantly in the cytoplasm of theEhrlichiae. The localization of gp19 is in contrast to another E. canisglycoprotein (gp36) that we reported to be differentially expressedprimarily on the surface of dense-cored cells (Doyle et al., 2006).However, similar to the gp36, the gp19 was also observed extracellularlyin the morula fibrillar matrix, and associated with the morula membrane.The expression of gp19 on Ehrlichiae, fibrillar matrix and the morulamembrane was further corroborated with immunofluorescence using dualstaining with Dsb, which is not secreted and is present of bothreticulate and dense cored organisms (McBride et al., 2002). Some smallmorulae appeared to have less gp19, suggesting that expression of gp19becomes more predominant as the morula matures. The E. canis gp19 doesnot have an amino-terminal signal sequence; therefore, the export ofthis protein probably involves a sec-independent secretion system (Type1 or Type III).

The E. canis gp19 was highly conserved in E. canis isolates examinedfrom the United States, Mexico and Brazil. The conservation of majorimmunoreactive genes (p28, gp140, gp36) in geographically separated E.canis isolates has been consistently reported (Doyle et al., 2006;McBride et al., 2000; McBride et al., 1999; Ndip et al., 2005; Yu etal., 2000). This indicates that globally effective vaccines and reliableimmunodiagnostics for E. canis based on major immunoreactive proteinssuch as the gp19 are feasible.

Example 4 Enhanced Sensitivity and Species-Specific Immunodiagnosis ofEhrlichia canis Infection by Enzyme-Linked Immunosorbent Assay withConserved Immunoreactive Glycoproteins gp36 and gp19

Ehrlichia canis is the primary etiologic agent of canine monocyticehrlichiosis (CME), a globally distributed and potentially fatal diseaseof dogs. The inventor previously reported the identification of twoconserved major immunoreactive antigens, gp36 and gp19, the firstproteins to elicit an E. canis-specific antibody response, and the gp200and p28, which elicit strong antibody responses later in the acuteinfection. In the present invention, the sensitivity and specificity offive recombinant E. canis proteins were evaluated for immunodiagnosis ofE. canis infection using an enzyme-linked immunosorbent assay (ELISA).Recombinant gp36, gp19 and gp200 polypeptides (N and C) exhibited 100%sensitivity and specificity compared with IFA (gold standard) indetecting antibodies in dogs that were naturally infected with E. canis.Furthermore, enhanced sensitivity of gp36 and gp19 compared to IFA wasdemonstrated with experimentally infected E. canis dogs, in whichantibodies were detected as much as 2 weeks earlier, on day 14 postinoculation. In addition, the gp36 and gp19 were not cross-reactive withantibodies in sera from E. chaffeensis-infected dogs, and thus providedspecies-specific serologic discrimination between E. canis and E.chaffeensis infections. This is the first study to demonstrate improveddetection capability with recombinant protein technology compared to the“gold standard” IFA, and may eliminate the remaining obstaclesassociated with immunodiagnosis of E. canis infections, includingspecies-specific identification and lack of sensitivity associated withlow antibody titers that occur early in the acute infection.

Materials and Methods

Experimental animals. Dogs and protocols used in experimental E. canisinfections were previously described (McBride et al., 2003). Forexperimental E. chaffeensis infections, two one-year old healthy beagleswere obtained from a commercial source and housed at the University ofTexas Medical Branch Laboratory Animal Resources facility, which isaccredited by the American Association for the Accreditation ofLaboratory Animal Care. Prior to the study, dogs were demonstrated tolack abnormalities on physical examination and have no detectableantibodies to E. chaffeensis by IFA. The experimental protocol wasapproved by the Animal Care and Use Committee at the University of TexasMedical Branch.

E. chaffeensis and E. canis inocula. The tissue culture infectious dose(TCID) of the E. chaffeensis inoculum was determined by inoculation ofDH82 monolayers plated in 24 well tissue culture plates with 10-folddilutions (10⁻¹ to 10⁻⁵) of inoculum (0.2 ml) in MEM. The inoculum wasincubated for 1 hr at 37° C. followed by the addition of 1 ml of growthmedium. Seven days after inoculation, the TCID was determined byidentification of E. chaffeensis in inoculated cells by IFA. The TCID ofthe E. canis inoculum was determined as previously described (Gaunt etal., 1996).

Experimental E. canis and E. chaffeensis infection in dogs. Two dogswere experimentally infected with E. chaffeensis (Arkansas strain)propagated in a mouse embryo cell line as previously described (Chen etal., 1995). Infected cells from six T-150 flasks were harvested bycentrifugation at 13,000×g, for 25 min after the cells were 80%infected. Two dogs received 4 ml of E. chaffeensis cell suspensionintravenously immediately after preparation, and the TCID50 wasdetermined retrospectively. Immune serum was collected four weeks afterinoculation, and anti-E. chaffeensis and anti-E. canis antibody titersdetermined by IFA. Fifteen dogs were experimentally infected with E.canis, and serum collected at weekly intervals as previously described(McBride et al., 2003).

Dog sera. Serum samples from ill dogs exhibiting clinical signs orhematologic abnormalities consistent with CME were submitted to theLouisiana Veterinary Medical Diagnostic Laboratory (LAVMDL) fromveterinarians statewide as previously described (McBride et al., 2001).Sera were screened by IFA (1:40) and separated into groups as Ehrlichiapositive and negative sera. Sera from healthy dogs were obtained fromone-year old healthy beagles from a commercial breeder (Marshall Farms,N.Y.).

Cloning of the genes of E. canis recombinant proteins. The gp19 (nt7-411), gp36 (nt 28-816), gp200N (nt 22-564) and gp200C (nt 3665-4188)and p28-3 gene (nt 82-695) were cloned into prokaryotic expressionvectors as previously described (Doyle et al., 2006; McBride et al.,2006; McBride et al., 2001; McBride et al., 2000; Nethery et al., 2006).The primers were designed for in-frame insertion of amplicons into thepUni/V5-His-TOPO vector and recombined with pBAD Thio-E Echo acceptorvector (p28) (Invitrogen Corporation, Carlsbad, Calif.) or cloneddirectly into a pBAD/TOPO ThioFusion expression vector (gp19, gp36,gp200N and gp200C) (Invitrogen).

Expression and purification of E. canis recombinant proteins. The gp19,gp36, gp200N-terminal and gp200C-terminal recombinant proteins wereexpressed in E. coli (TOP10) after induction with 0.02% of arabinose for2 hr. Bacteria (from 10 L of fermentation culture) were harvested bycentrifugation at 5,000×g for 40 min and resuspended in PBS. Recombinantproteins (gp19, gp36, gp200N and gp200C) were purified under nativeconditions by lysing the bacteria resuspended in lysis buffer (PBS,0.05% Triton 100×, 0.5 M NaCl, 1 mM PMSF and 5 mM imidazole) anddisrupted using a French Press at 1100 psi in ice water and pelletingthe insoluble material by centrifugation at 10,000×g for 1 hr. Theclarified supernatant was loaded onto an equilibrated Ni-NTA column (50ml column). The bound recombinant protein was washed with 15 columnvolumes of increasing concentrations of imidazole (4%, 8%, 20% and 100%)and eluted with 250 mM imidazole in lysis buffer. Recombinant p28protein was purified under denaturing conditions by sonicating thepelleted bacteria resuspended in lysis buffer (50 mM Tris-HCl, 400 mMNaCl, 1 mM PMSF and 0.1% Triton 100×) at 50 W for 30 min (20 s on, 20 soff) in ice water and pelleting the insoluble material by centrifugation(10,000×g) for 30 min. The pellet was washed three times first with 2 Murea, then 4 M urea in lysis buffer and then with water stirring for 30min at room temperature and pelleted (6000 g) for 30 min. The final washwas performed in 4 M urea plus 1% Triton X100 and 0.1% deoxycholic acidwith stirring for 1 hr at room temperature and pelleted bycentrifugation (10,000×g, 45 min). The pellet was resuspended in samplebuffer (4 M Urea, 6 M guanidine and 50 mM 2-mercaptoethanol) withovernight stirring at 4° C. and pelleted (10,000×g, 40 min). Theclarified supernatant was loaded onto an equilibrated reversed phasecolumn (26/10 XK, Amersham Biosciences), washed with buffer A (0.1% TFA)and eluted with 6 column volumes of increasing ratios (from 0% to 100%of buffer B) of buffer A and buffer B (0.1% TFA and 85% acetonitrile).

Enzyme-linked immunosorbent assay (ELISA). Antibody response to five E.canis recombinant proteins (gp36, gp19, p28, gp200N and gp200C) wasevaluated by an enzyme-linked immunosorbant assay (ELISA). The ELISAprotocol was optimized including choice of ELISA plate, proteinconcentrations, serum dilutions, and blocking buffers. The recombinantgp36 (0.3 μg/ml), gp19 (1.2 mg/ml), p28 (2.5 μg/ml), gp200N (1.4 μg/ml),gp200C (0.5 μg/ml), and thioredoxin control (2.5 μg/ml) were diluted inPBS and assay plate (Nunc-Immuno™ Plates with Polysorp™ Surface, NUNC,Roskilde, Denmark) wells were coated with 50 μl containing therecombinant proteins and incubated at room temperature for 2 hr orovernight at 4° C. The plates were washed 4 times with 200 μl of washbuffer (PBS and Tween 20, 0.05%), and blocked with 100 μl of blockingbuffer (10% equine serum in PBS; HyClone Laboratories, Inc., Logan,Utah) and incubated for 1 hr at 37° C. Each primary antibody was diluted1:250 in blocking buffer and 50 μl of the antibody was added toduplicate test wells containing antigen, a control well containingrecombinant thioredoxin (negative control), and a blank well containingno antigen and incubated at room temperature for 1 hr. The plates werewashed, and 50 μl of affinity-purified peroxidase labeled goat anti-dogIgG (H & L) (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) diluted1:1000 in blocking buffer was added to each well. The plates wereincubated for 1 hr at room temperature and washed. Bound antibody wasdetected after addition of substrate (100 μl) (Sure Blue Reserveperoxidase substrate, Kirkegaard & Perry Laboratories). Plates were readon a tunable microplate reader (Molecular Devices, Sunnyvale. Calif.) atA630 after incubation at room temperature for 20 min. The absorbance ofeach sample was plotted as the optical density at 630 nm (OD630), andthe background from the negative control well was subtracted from eachcorresponding sample to determine the final absorbance.

Gel electrophoresis and Western immunoblotting. E. canis recombinantproteins were separated by sodium dodecyl-sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, andWestern immunoblots were performed as previously described (McBride etal., 2003).

IFA. Antibody status of dogs experimentally infected with E. canis,clinically ill and naturally infected dogs was determined as previouslydescribed (McBride et al., 2003). Antibody status of healthy dogs and E.chaffeensis experimentally and naturally infected dogs was performedsimilarly with E. canis (Jake strain) and E. chaffeensis (Arkansasstrain) antigen slides. Sera were assayed using two-fold dilutions inPBS starting at 1:64.

Comparison of Antibody Kinetics Against E. canis Recombinant Proteins byWestern Blot and ELISA

Antibodies to the E. canis major immunoreactive proteins developdifferentially during the acute infection (McBride et al., 2003). Theantibody response to E. canis recombinant proteins in threeexperimentally infected dogs was examined by ELISA and Westernimmunoblot to determine the correlation between the two immunoassays andto determine if kinetics previously observed with native E. canislysates were reproduced with the recombinant proteins. Antibodies insera from the three E. canis-infected dogs reacted earliest (day 14)with the recombinant gp36 by both Western immunoblot and ELISA, followedby the gp19 (day 21). The p28 and gp200 N- and C-terminal polypeptidesexhibited similar detection sensitivity, reacting with antibodies laterin the course of infection (days 28 to 35) approximately two weeks laterthan the gp36 (FIG. 9).

Analytical Sensitivity and Specificity of E. canis RecombinantGlycoprotein ELISA

The current “gold standard” for immunodiagnosis is the indirectfluorescent antibody (IFA) test. This standard was used to determine thesensitivity and specificity of our recombinant protein ELISA. Antibodiesagainst recombinant gp36, gp19, gp200N and gp200C were detected in all29 positive IFA samples by ELISA from experimentally (range 1280to >10,240) and naturally (antibody titers: 4, ≧3200; 4, 1600; 3, 800;and 2, 400) infected dogs with E. canis (Table 3). The recombinantproteins (gp36, gp19 and gp200 N- and C-terminal) exhibited 100%specificity compared to IFA with sera from healthy and ill dogs (Table3). Conversely, recombinant p28 exhibited high levels of nonspecificantibody binding (above negative control levels) with some dog sera andthus had a substantially lower specificity (60%).

TABLE 3 Analytical sensitivity and specificity of E. canisimmunodiagnosis with recombinant proteins and IFA Dogs (%) withdetectable antibodies IFA gp36 gp19 gp200N gp200C ExperimentallyInfected E. canis (n = 15)* 15 (100) 15 (100) 15 (100) 15 (100) 15 (100)Naturally Infected E. canis (n = 14) 14 (100) 14 (100) 14 (100) 14 (100)14 (100) Total (n = 29) 29 (100) 29 (100) 29 (100) 29 (100) 29 (100)Clinically Healthy (n = 10) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Clinically Ill(n = 26) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Total (n = 36) 0 (0) 0 (0) 0 (0)0 (0) 0 (0) Experimentally Infected E. chaffeensis (n = 2) 2 (100) 0 (0)0 (0) 0 (0) 0 (0) Naturally Infected E. chaffeensis (n = 2)** 2 (100) 0(0) 0 (0) 0 (0) 0 (0)

Earlier detection of anti-E. canis antibodies with recombinantglycoproteins. Dogs experimentally infected with E. canis (n=15), inwhich antibody response kinetics were defined (McBride et al., 2003),were used to determine detection sensitivity of IFA compared torecombinant proteins in ELISA. The experimentally infected dogs (1exception) developed IgG antibodies to E. canis gp36 that could bedetected by ELISA by 14 days post inoculation (dpi), and one third ofthese dogs (n=5) had antibodies that reacted with gp19 (Table 3).Conversely, none of the dogs had detectable IgG antibodies by IFA on 14dpi (Table 3). Antibodies were detectable by IFA in only four dogs at 21dpi. The IFA and recombinant glycoprotein ELISA sensitivity becamecomparable at 28 dpi, but complete agreement was not attained until 42dpi (Table 4).

TABLE 4 Comparison of serologic detection sensitivity of E. canisinfection by IFA and ELISA (gp36 and gp19) in experimentally infecteddogs Day 0 Day 7 Day 14 Day 21 IFA gp36 gp19 IFA gp36 gp19 IFA gp36 gp19IFA gp36 gp19 32 − − − − − − − + + + + + 33 − − − − − − − + − + + + 34 −− − − − − − + − + + + 35 − − − − − − − + + − + + 37 − − − − − − − + +− + + 41 − − − − − − − + − − + + 43 − − − − − − − + − − + + 44 − − − − −− − + − + + + 45 − − − − − − − + + − + − 46 − − − − − − − + − − + − 48 −− − − − − − + + − + + 51 − − − − − − − − − − − − 52 − − − − − − − + −− + + 54 − − − − − − − + − − + − 59 − − − − − − − + − − + − Day 28 Day35 Day 42 IFA gp36 gp19 IFA gp36 gp19 IFA gp36 gp19 32 + + + + + + + + +33 + + + + + + + + + 34 + + + + + + + + + 35 + + + + + + + + +37 + + + + + + + + + 41 + + + + + + + + + 43 + + + + + + + + +44 + + + + + + + + + 45 + + + + + + + + + 46 + + + + + + + + + 48 − − −− + − + + + 51 − + − + + + + + + 52 + + + + + + + + +54 + + + + + + + + + 59 − + − + + + + + +

Species-specific immunodiagnosis with gp19 and gp36. Four dogs infectedwith E. chaffeensis (2-experimental and 2-natural) were IFA positive toE. canis antigen, but did not react with the E. canis recombinantproteins (gp36, gp19 and gp200). The anti-E. chaffeensis antibody titersin sera from dogs experimentally infected with E. chaffeensis were≧1280. The anti-E. chaffeensis IFA antibody titers of the dogs naturallyinfected with E. chaffeensis were 1:400 and 1:800 to homologous antigen(1:64 to E. canis).

SIGNIFICANCE OF THE PRESENT EMBODIMENT

Early diagnosis of CME in the acute stage of infection followed bytreatment with doxycycline ensures the best prognosis. Detection of E.canis antibodies by IFA is currently the most widely used method fordiagnosis of CME and is considered the “gold standard” (Waner et al.,2001). However, IFA is routinely performed in large reference diagnosticlaboratories and is not useful as a point-of-care diagnostic orscreening test because requires a high level of technical experience, issubject to inter- and intralaboratory variation and misinterpretation,and requires expensive fluorescent microscopy equipment. Furthermore,the IFA uses E. canis-infected cells that are not well defined and thatcontain antigens (heat shock and p28/p30) capable of reacting withantibodies generated against other genus members (E. chaffeensis and E.ewingii) and organisms from other genera (Neorickettsia) (Comer et al.,1999). Thus, the possibility of multiple tick-borne infections in dogscomplicates serological diagnosis by IFA (Kordick et al., 1999).Currently, point-of-care diagnostic tests (Snap 3Dx, IDEXX Laboratories;Dip-S-Tick, PanBio InDx; Immunocomb, Biogal) that are commerciallyavailable utilize whole cell antigen or synthetic or recombinantproteins/peptides from two major outer membrane proteins (p30 andp30-1). The Snap 3Dx assay appears to be one of the most widely usedtests, but two recent studies have concluded that sensitivity appears tobe substantially less than IFA (Belanger et al., 2002; Harrus et al.,2002), a problem that is more pronounced with sera containing low (<320)antibody titers (Harrus et al., 2002; O'Connor et al., 2006).Furthermore, all of the commercially available assays are unable todifferentiate between various Ehrlichia spp. that are known to causeinfections in dogs. Hence, immunodiagnostics capable of providing bettersensitivity, particularly during early acute infection, and the abilityto differentiate the specific agent responsible for the infection;utilizing a well characterized and consistently reproducible recombinantor synthetic antigen are needed, but unavailable.

The recent molecular identification of several distinct but conservedmajor immunoreactive proteins of E. canis including gp36, gp19, gp200and p28/30 has created new opportunities for substantial improvements inserologic diagnosis of CME (Doyle et al., 2006; McBride et al., 2003;McBride et al., 2001; McBride et al., 1999; Ohashi et al., 1998). Theinventor has previously reported that the E. canis gp36, gp19 and gp200are molecularly and immunologically distinct from the respectiveorthologs in E. chaffeensis (gp47, VLPT and gp200), and that two ofthese characterized proteins (gp36 and gp19) are the first to elicit anantibody response in E. canis-infected dogs (McBride et al., 2003). Inaddition, these proteins are conserved among geographically dispersed E.canis strains (Doyle et al., 2006; McBride et al., 2006; McBride et al.,2000; McBride et al., 1999). Therefore, these antigens have a highpotential to facilitate the development of ultrasensitive and highlyspecific new generation immunodiagnostics for detection of E. canisinfection. It was considered that in certain embodiments these proteinswould provide increased sensitivity over whole cell antigen (IFA) fordetecting antibodies early in the infection. In this invention, it wasdemonstrated that two recombinant E. canis proteins (gp36 and gp19) usedin an ELISA format provided enhanced sensitivity compared to IFA fordetecting antibodies during the early immune response and were highlyspecific for E. canis.

The molecularly characterized recombinant proteins (gp36, gp19, p28/30and gp200) reacted with antibody from infected canine sera with kineticssimilar to that reported with corresponding proteins in native E. canislysates (McBride et al., 2003) in two immunoassay formats (ELISA andmembrane) that are commonly used for point-of-care diagnostic tests.These results confirm that the recombinant proteins are suitablesurrogates for native ehrlichial proteins and react similarly withantibodies generated during an infection. Furthermore, consistentresults obtained by two immunoassay formats indicate that these proteinscould provide consistent sensitivity regardless of the assay formatutilized. In this particular study, Western immunoblotting providedsimilar results as compared to the ELISA, but results can belaboratory-dependent, and this technique is laborious, time consumingand not well suited for point-of-care tests.

The analytical sensitivity of the E. canis recombinant proteinscompletely correlated with the IFA using sera from dogs with natural andexperimental infections. The inventors previously reported 100%sensitivity with E. canis gp200-N (P43), and those results wereconfirmed in this study (McBride et al., 2001). However, it was recentlyidentified that there are five major epitopes within the gp200 protein(Nethery et al., 2006). The gp200-N (P43) contains a single majorepitope and carboxy-terminal region, gp200-C, contains two majorantibody epitopes (Nethery et al., 2006). The antibody response to bothgp200 recombinant proteins developed later than the gp36 and gp19, butthey reacted strongly with antibody in late acute phase serum fromexperimental dogs. These findings were consistent with previousinvestigations in which there was observed a strong late acute phaseantibody response to the gp200 (McBride et al., 2003; McBride et al.,2001). Antibody to the E. canis P28 also developed later in the lateacute phase immune response. It was previously reported similar antibodyresponse kinetics that were consistent with both native and recombinantP28 (McBride et al., 2003). Some nonspecific responses to P28 wereobserved in the ELISA format, but this is due to other contaminatingproteins, in specific embodiments. The P28 is very insoluble thusproducing a highly purified recombinant protein is very difficult toachieve. Nevertheless, results obtained by Western immunoblotting inthis study and other studies suggest that highly purified p28/p30 is aspecific immunodiagnostic antigen (Belanger et al., 2002; McBride etal., 2003; McBride et al., 2001).

The first detectable antibodies to E. canis are directed at the gp36 andgp19 (Doyle et al., 2006; McBride et al., 2003). All of the E. canisrecombinant proteins provided sensitivity similar to IFA in naturallyinfected dogs; however, in the experimentally infected group of dogswhere the kinetics of the antibody response could be accuratelydetermined, the gp36 and gp19 detected antibodies 7 to 14 days earlierthan IFA or ELISA using the gp200 and p28. To our knowledge, this is thefirst demonstration that species-specific E. canis proteins are moresensitive than whole cell antigen for detection of low antibody levelsproduced during early acute ehrlichial infections. Many E. canisproteins may be suitable for detecting late acute phase antibodies, andsensitivities of specific proteins appear to be related to the diseasephase. The sensitivity of E. canis antigens such as p28/p30, gp200 andMAP2 for detecting antibodies appears to be best in a later diseasephase when sera contain medium to high levels of antibody. However, serawith low antibody levels, such as those obtained early in the infection,pose more difficulties with these recombinant antigens and whole cellantigen (Harms et al., 2002; O'Connor et al., 2006). This situation maybe particularly relevant to sera collected from dogs early in theinfection when antibody levels are low, and when an accurate diagnosiscan be most challenging serologically.

The gp36 and gp19 have species-specific serine-rich major epitopes thathave been identified and molecularly characterized (Doyle et al., 2006;McBride et al., 2006). Likewise, the E. canis and E. chaffeensis gp200orthologs are antigenically distinct and have epitopes that have beenmolecularly characterized (McBride et al., 2003; McBride et al., 2001;Nethery et al., 2006). The major epitopes on gp36, gp19, and gp200appear to have carbohydrate immunodeterminants that contribute to theimmunoreactivity of the epitopes (Doyle et al., 2006; McBride et al.,2006; Nethery et al., 2006). These major immunoreactive antigens candiscriminate serologically between E. canis and the most closely relatedorganism, E. chaffeensis, and will enable the development of highlyspecific assays capable of discrimination of the specific infectingagent. Another major immunoreactive antigen (gp120) of E. chaffeensiscapable of sensitive species-specific discrimination has also beenreported (Yu et al., 1996; Yu et al., 1997; Yu et al., 1999). Thus,highly defined recombinant antigens that include the major epitopes ofthe E. canis gp36 and/or gp19 and E. chaffeensis gp120 could be utilizedin the same assay for specific diagnosis of E. canis and E. chaffeensisinfections.

The reliability of serologic diagnosis of infections with recombinant orsynthetic antigens depends on the lack of antigenic variability of theantigen that is selected. In the case of E. canis, many of the majorimmunoreactive antigens, including gp36, gp19, gp200 and p28 that havethe potential to be utilized for serodiagnosis, are highly conserved ingeographically distinct isolates (Doyle et al., 2006; McBride et al.,2006; McBride et al., 2000; McBride et al., 1999; Yu et al., 2000).Conversely, E. chaffeensis exhibits more diversity among differentisolates, but the antibody epitope of the gp120 appears to be wellconserved (Chen et al., 1997; Doyle et al., 2006; Reddy and Streck,2000; Doyle et al., 2006; Yu et al., 1997; Yu et al., 2000). Moreover,differential expression of the major outer membrane proteins (p28/p30),which have antigenically distinct hypervariable regions that containantibody epitopes (Barnewall et al., 1999; Li et al., 2002; Li et al.,2002; McBride et al., 1999; Ohashi et al., 1998; Ohashi et al., 1998;Unver et al., 2002; Yu et al., 2000) may also contribute to variationsin serologic responses to E. canis and E. chaffeensis. Thus, it wasconcluded that antigens such as the gp36 and gp19 that are highlyconserved single gene proteins that minimize or eliminate potential forserologic variability have the best potential for development ofglobally useful ultrasensitive and species-specific immunodiagnosticsthat overcome these obstacles associated with CME serodiagnosis.

Example 5 Vaccines of the Invention

In particular aspects of the invention, the immunogenic compositions ofthe present invention are suitable as a vaccine, such as a subunitvaccine. In other aspects of the invention, the immunogenic compositionsare referred to as immunoprotective.

Specifically, one or more compositions of the invention, such as thosecomprising an E. canis gp19 epitope, for example, are administered to amammal, such as a human, canine, bovine, or equine animal, for example.Serum from the mammal may be assayed for an immune response, such as bydetecting antibodies in the serum. The mammal is then subjected tosubsequent challenge with the pathogenic organism, such as the E. canisorganism, or another appropriate composition, and immunoprotection isdetermined. Controls may be employed, such as immunization with, forexample, a mutated epitope or an epitope that does not comprise acarbohydrate moiety. Complete or partial protection against thesubsequent challenge demonstrates the immunoprotective nature of thecomposition, and the composition is a vaccine. Partial protection may bedefined as protecting from developing or delaying from developing atleast one symptom of the infection or protecting from at least onesymptom becoming worse.

REFERENCES

All patents and publications mentioned in the specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

PATENTS AND PATENT APPLICATIONS

U.S. Pat. No. 5,440,013

U.S. Pat. No. 5,618,914

U.S. Pat. No. 5,670,155

U.S. Pat. No. 5,446,128

U.S. Pat. No. 5,710,245

U.S. Pat. No. 5,840,833

U.S. Pat. No. 5,859,184

U.S. Pat. No. 5,929,237

U.S. Pat. No. 5,475,085

U.S. Pat. No. 5,672,681

U.S. Pat. No. 5,674,976

U.S. Pat. No. 4,554,101

PUBLICATIONS

-   Alleman, A. R., A. F. Barbet, M. V. Bowie, H. L. Sorenson, S. J.    Wong, and M. Belanger. 2000. Expression of a gene encoding the major    antigenic protein 2 homolog of Ehrlichia chaffeensis and potential    application for serodiagnosis. J. Clin. Microbiol. 38:3705-3709.-   Alleman, A. R., L. J. McSherry, A. F. Barbet, E. B.    Breitschwerdt, H. L. Sorenson, M. V. Bowie, and M. Belanger. 2001.    Recombinant major antigenic protein 2 of Ehrlichia canis: a    potential diagnostic tool. J. Clin. Microbiol. 39:2494-2499.-   Barnewall, R. E., N. Ohashi, and Y. Rikihisa. 1999. Ehrlichia    chaffeensis and E. sennetsu, but not the human granulocytic    erhlichiosis agent, colocalize with transferrin receptor and    up-regulate transferrin receptor mRNA by activating iron-responsive    protein 1. Infect. Immun. 67:2258-2265.-   Belanger, M., H. L. Sorenson, M. K. France, M. V. Bowie, A. F.    Barbet, E. B. Breitschwerdt, and A. R. Alleman. 2002. Comparison of    serological detection methods for diagnosis of Ehrlichia canis    infections in dogs. J Clin Microbiol. 40:3506-3508.-   Benson, G. 1999. Tandem repeats finder: a program to analyze DNA    sequences. Nucleic Acids Res. 27:573-580.-   Breitschwerdt, E. B., B. C. Hegarty, and S. I. Hancock. 1998.    Sequential evaluation of dogs naturally infected with Ehrlichia    canis, Ehrlichia chaffeensis, Ehrlichia equi, Ehrlichia ewingii, or    Bartonella vinsonii. J. Clin. Microbiol. 36:2645-2651.-   Bzymek, M. and S. T. Lovett. 2001. Instability of repetitive DNA    sequences: the role of replication in multiple mechanisms. Proc.    Natl. Acad. Sci. U.S.A 98:8319-8325.-   Chen, S. M., V. L. Popov, H. M. Feng, J. Wen, and D. H.    Walker. 1995. Cultivation of Ehrlichia chaffeensis in mouse embryo,    Vero, BGM, and L929 cells and study of Ehrlichia-induced cytopathic    effect and plaque formation. Infect. Immun. 63:647-655.-   Chen, S. M., X. J. Yu, V. L. Popov, E. L. Westerman, F. G. Hamilton,    and D. H. Walker. 1997. Genetic and antigenic diversity of Ehrlichia    chaffeensis: comparative analysis of a novel human strain from    Oklahoma and previously isolated strains. J. Infect. Dis.    175:856-863.-   Codner, E. C. and L. L. Farris-Smith. 1986. Characterization of the    subclinical phase of ehrlichiosis in dogs. J. Am. Vet. Med. Assoc.    189:47-50.-   Collins, N. E., J. Liebenberg, E. P. de Villiers, K. A. Brayton, E.    Louw, A. Pretorius, F. E. Faber, H. H. van, A. Josemans, K. M.    van, H. C. Steyn, M. F. van Strijp, E. Zweygarth, F. Jongejan, J. C.    Maillard, D. Berthier, M. Botha, F. Joubert, C. H. Corton, N. R.    Thomson, M. T. Allsopp, and B. A. Allsopp. 2005. The genome of the    heartwater agent Ehrlichia ruminantium contains multiple tandem    repeats of actively variable copy number. Proc. Natl. Acad. Sci.    U.S.A 102:838-843.-   Corner, J. A., W. L. Nicholson, J. G. Olson, and J. E. Childs. 1999.    Serologic testing for human granulocytic ehrlichiosis at a national    referral center. J. Clin. Microbiol. 37:558-564.-   Cowell, R. L., R. D. Tyler, K. D. Clinkenbeard, and J. H.    Meinkoth. 1988. Ehrlichiosis and polyarthritis in three dogs. J. Am.    Vet. Med. Assoc. 192:1093-1095.-   Dawson, J. E. and S. A. Ewing. 1992. Susceptibility of dogs to    infection with Ehrlichia chaffeensis, causative agent of human    ehrlichiosis. Am. J. Vet. Res. 53:1322-1327.-   Dawson, J. E., K. L. Biggie, C. K. Warner, K. Cookson, S.    Jenkins, J. F. Levine, and J. G. Olson. 1996. Polymerase chain    reaction evidence of Ehrlichia chaffeensis, an etiologic agent of    human ehrlichiosis, in dogs from southeast Virginia. Am. J. Vet.    Res. 57:1175-1179.-   Doyle, C. K., A. M. Cardenas, D. M. Aguiar, M. B. Labruna, L. M.    Ndip, X. J. Yu, and McBride J. W. 2006. Molecular characterization    of E. canis gp36 and E. chaffeensis gp47 tandem repeats among    different geographic locations. Ann. N.Y. Acad. Sci. 1063.-   Doyle, C. K., K. A. Nethery, V. L. Popov, and J. W. McBride. 2006.    Differentially expressed and secreted major immunoreactive protein    orthologs of Ehrlichia canis and E. chaffeensis elicit early    antibody responses to epitopes on glycosylated tandem repeats.    Infect. Immun. 74:711-720.-   Doyle, C. K., M. B. Labruna, E. B. Breitschwerdt, Y. W. Tang, R. E.    Corstvet, B. C. Hegarty, K. C. Bloch, P. Li, D. H. Walker, and J. W.    McBride. 2005. Detection of medically important Ehrlichia spp. by    quantitative multicolor Taqman real-time PCR of the dsb gene. J.    Mol. Diagn. 10:504-510.-   Doyle, C. K., X. Zhang, V. L. Popov, and J. W. McBride. 2005. An    immunoreactive 38-kilodalton protein of Ehrlichia canis shares    structural homology and iron-binding capacity with the ferric    ion-binding protein family. Infect. Immun. 73:62-69.-   Dunning Hotopp, J. C., M. Lin, R. Madupu, J. Crabtree, S. V.    Angiuoli, J. Eisen, R. Seshadri, Q. Ren, M. Wu, T. R. Utterback, S.    Smith, M. Lewis, H. Khouri, C. Zhang, H. Niu, Q. Lin, N. Ohashi, N.    Zhi, W. Nelson, L. M. Brinkac, R. J. Dodson, M. J. Rosovitz, J.    Sundaram, S. C. Daugherty, T. Davidsen, A. S. Durkin, M.    Gwinn, D. H. Haft, J. D. Selengut, S. A. Sullivan, N. Zafar, L.    Zhou, F. Benahmed, H. Forberger, R. Halpin, S. Mulligan, J.    Robinson, O. White, Y. Rikihisa, and H. Tettelin. 2006. Comparative    genomics of emerging human ehrlichiosis agents. PLoS Genet. 2:e21.

Frutos, R., A. Viari, C. Ferraz, A. Morgat, S. Eychenie, Y. Kandassamy,I. Chantal, A. Bensaid, E. Coissac, N. Vachiery, J. Demaille, and D.Martinez. 2006. Comparative genomic analysis of three strains ofEhrlichia ruminantium reveals an active process of genome sizeplasticity. J Bacteriol 188:2533-2542.

-   Gaunt, S. D., R. E. Corstvet, C. M. Berry, and B. Brennan. 1996.    Isolation of Ehrlichia canis from dogs following subcutaneous    inoculation. J. Clin. Microbiol. 34:1429-1432.-   Goldman, E. E., E. B. Breitschwerdt, C. B. Grindem, B. C.    Hegarty, J. J. Walls, and J. S. Dumler. 1998. Granulocytic    ehrlichiosis in dogs from North Carolina and Virginia. J Vet Intern    Med 12:61-70.-   Harrus, S., A. R. Alleman, H. Bark, S. M. Mahan, and T. Waner. 2002.    Comparison of three enzyme-linked immunosorbant assays with the    indirect immunofluorescent antibody test for the diagnosis of canine    infection with Ehrlichia canis. Vet. Microbiol. 86:361-368.-   Hinc, K., K. Nagorska, A. Iwanicki, G. Wegrzyn, S. J. Seror, and M.    Obuchowski. 2006. Expression of genes coding for GerA and GerK spore    germination receptors is dependent on the protein phosphatase PrpE.    J Bacteriol 188:4373-4383.-   Johannesson et al., 1999, “Bicyclic tripeptide mimetics with reverse    turn inducing properties.” J. Med. Chem. 42:601-608.-   Julenius, K., A. Molgaard, R. Gupta, and S. Brunak. 2005.    Prediction, conservation analysis, and structural characterization    of mammalian mucin-type O-glycosylation sites. Glycobiology    15:153-164.-   Knowles, T. T., A. R. Alleman, H. L. Sorenson, D. C. Marciano, E. B.    Breitschwerdt, S. Harrus, A. F. Barbet, and M. Belanger. 2003.    Characterization of the major antigenic protein 2 of Ehrlichia canis    and Ehrlichia chaffeensis and its application for serodiagnosis of    ehrlichiosis. Clin. Diagn. Lab Immunol. 10:520-524.-   Kordick, S. K., E. B. Breitschwerdt, B. C. Hegarty, K. L.    Southwick, C. M. Colitz, S. I. Hancock, J. M. Bradley, R.    Rumbough, J. T. Mcpherson, and J. N. MacCormack. 1999. Coinfection    with multiple tick-borne pathogens in a Walker hound kennel in North    Carolina. J. Clin. Microbiol. 37:2631-2638.-   Kuehn, N. F. and S. D. Gaunt. 1985. Clinical and hematologic    findings in canine ehrlichiosis. J. Am. Vet. Med. Assoc.    186:355-358.-   Levine, A., F. Vannier, C. Absalon, L. Kuhn, P. Jackson, E.    Scrivener, V. Labas, J. Vinh, P. Courtney, J. Garin, and S. J.    Seror. 2006. Analysis of the dynamic Bacillus subtilis Ser/Thr/Tyr    phosphoproteome implicated in a wide variety of cellular processes.    Proteomics. 6:2157-2173.-   Li, J. S., E. Yager, M. Reilly, C. Freeman, G. R. Reddy, A. A.    Reilly, F. K. Chu, and G. M. Winslow. 2001. Outer membrane    protein-specific monoclonal antibodies protect SCID mice from fatal    infection by the obligate intracellular bacterial pathogen Ehrlichia    chaffeensis. J. Immunol. 166:1855-1862.-   Li, J. S., F. Chu, A. Reilly, and G. M. Winslow. 2002. Antibodies    highly effective in SCID mice during infection by the intracellular    bacterium Ehrlichia chaffeensis are of picomolar affinity and    exhibit preferential epitope and isotype utilization. J. Immunol.    169:1419-1425.-   Madec, E., A. Laszkiewicz, A. Iwanicki, M. Obuchowski, and S.    Seror. 2002. Characterization of a membrane-linked Ser/Thr protein    kinase in Bacillus subtilis, implicated in developmental processes.    Mol. Microbiol. 46:571-586.-   Mahan, S. M., T. C. McGuire, S. M. Semu, M. V. Bowie, F.    Jongejan, F. R. Rurangirwa, and A. F. Barbet. 1994. Molecular    cloning of a gene encoding the immunogenic 21 kDa protein of Cowdria    ruminantium. Microbiol. 140 (Pt 8):2135-2142.-   Mavromatis, K., C. K. Doyle, A. Lykidis, N. Ivanova, M. P.    Francino, P. Chain, M. Shin, S. Malfatti, F. Larimer, A.    Copeland, J. C. Detter, M. Land, P. M. Richardson, X. J. Yu, D. H.    Walker, J. W. McBride, and N. C. Kyrpides. 2006. The genome of the    obligately intracellular bacterium Ehrlichia canis reveals themes of    complex membrane structure and immune evasion strategies. J    Bacteriol 188:4015-4023.-   McBride J. W., C. K. Doyle, X. F. Zhang, A. M. Cardenas, V. L.    Popov, K. A. Nethery, and M. E. Woods. 2006. Ehrlichia canis 19-kDa    glycoprotein ortholog of E. chaffeensis variable length PCR target    contains a single serine-rich epitope defined by a carbohydrate    immunodetermiant. Infect. Immun.-   McBride J W, R. E. Corstvet, S. D. Gaunt, C. Boudreaux, T. Guedry,    and D. H. Walker. 2003. Kinetics of antibody response to Ehrlichia    canis immunoreactive proteins. Infect. Immun. 71:2516-2524.-   McBride J W, R. E. Corstvet, S. D. Gaunt, C. Boudreaux, T. Guedry,    and D. H. Walker. 2003. Kinetics of antibody response to Ehrlichia    canis immunoreactive proteins. Infect. Immun. 71:2516-2524.-   McBride, J. W., J. E. Comer, and D. H. Walker. 2003. Novel    immunoreactive glycoprotein orthologs of Ehrlichia spp. Ann. N.Y.    Acad. Sci. 990:678-684.-   McBride, J. W., L. M. Ndip, V. L. Popov, and D. H. Walker. 2002.    Identification and functional analysis of an immunoreactive    DsbA-like thio-disulfide oxidoreductase of Ehrlichia spp. Infect.    Immun. 70:2700-2703.-   McBride, J. W., R. E. Corstvet, E. B. Breitschwerdt, and D. H.    Walker. 2001. Immunodiagnosis of Ehrlichia canis infection with    recombinant proteins. J. Clin. Microbiol. 39:315-322.-   McBride, J. W., R. E. Corstvet, E. B. Breitschwerdt, and D. H.    Walker. 2001. Immunodiagnosis of Ehrlichia canis infection with    recombinant proteins. J. Clin. Microbiol. 39:315-322.-   McBride, J. W., X. J. Yu, and D. H. Walker. 1999. Molecular cloning    of the gene for a conserved major immunoreactive 28-kilodalton    protein of Ehrlichia canis: a potential serodiagnostic antigen.    Clin. Diag. Lab. Immunol. 6:392-399.-   McBride, J. W., X. J. Yu, and D. H. Walker. 2000. Glycosylation of    homologous immunodominant proteins of Ehrlichia chaffeensis and E.    canis. Infect. Immun. 68:13-18.-   McBride, J. W., X. Yu, and D. H. Walker. 2000. A conserved,    transcriptionally active p28 multigene locus of Ehrlichia canis.    Gene 254:245-252.-   Ndip, L. M., R. N. Ndip, S, N. Esemu, V. L. Dickmu, E. B.    Fokam, D. H. Walker, and J. W. McBride. 2005. ehrlichial infection    in Cameroonian canines by Ehrlichia canis and Ehrlichia ewingii.    Vet. Microbiol. 111:59-66.-   Nethery, K. A., C. K. Doyle, B. L. Elsom, N. K. Herzog, V. L. Popov,    and J. W. McBride. 2005. Ankyrin repeat containing immunoreactive    200 kD glycoprotein (gp200) orthologs of Ehrlichia chaffeensis and    Ehrlichia canis are translocated to the nuclei of infected    monocytes, p. 0-60. In 4th International Conference on Rickettsiae    and Rickettsial Diseases, Longrono, Spain.-   Nethery, K. A., Doyle C. K., X. F. Zhang, and McBride J. W. 2006.    Ehrlichia canis gp200 contains five major B cell epitopes defined by    O-linked carbohydrate immunodeterminants. Infect. Immun.-   Nielsen, H., J. Engelbrecht, S. Brunak, and G. von Heijne. 1997.    Identification of prokaryotic and eukaryotic signal peptides and    prediction of their cleavage sites. Protein Eng 10:1-6.-   Obuchowski, M., E. Madec, D. Delattre, G. Boel, A. Iwanicki, D.    Foulger, and S. J. Seror. 2000. Characterization of PrpC from    Bacillus subtilis, a member of the PPM phosphatase family. J    Bacteriol 182:5634-5638.-   O'Connor, T. P., Esty K. J., MacHenry P., and Hanscom J. L. 2002.    Performance evaluation of Ehrlichia canis and Borrelia burgdorferi    peptides in a new Dirofilaria immitis combinantion assay., p. 77-84.    In Recent advances in heartworm disease: symposium '01. American    Heartworm Society, Batavia, Ill.-   O'Connor, T. P., J. L. Hanscom, B. C. Hegarty, R. G. Groat,    and E. B. Breitschwerdt. 2006. Comparison of an indirect    immunofluorescence assay, western blot analysis, and a commercially    available ELISA for detection of Ehrlichia canis antibodies in    canine sera. Am. J. Vet. Res. 67:206-210.-   Ohashi, N., A. Unver, N. Zhi, and Y. Rikihisa. 1998. Cloning and    characterization of multigenes encoding the immunodominant    30-kilodalton major outer membrane proteins of Ehrlichia canis and    application of the recombinant protein for serodiagnosis. J. Clin.    Microbiol. 36:2671-2680.-   Ohashi, N., N. Thi, Y. Zhang, and Y. Rikihisa. 1998. Immunodominant    major outer membrane proteins of Ehrlichia chaffeensis are encoded    by a polymorphic multigene family. Infect. Immun. 66:132-139.-   Reddy, G. R. and C. P. Streck. 2000. Variability in the 28-kDa    surface antigen protein multigene locus of isolates of the emerging    disease agent Ehrlichia chaffeensis suggests that it plays a role in    immune evasion [published erratum appears in Mol Cell Biol Res    Commun 2000 January; 3(1):66]. Mol. Cell. Biol. Res. Commun.    1:167-175.-   Reddy, G. R., C. R. Sulsona, A. F. Barbet, S. M. Mahan, M. J.    Burridge, and A. R. Alleman. 1998. Molecular characterization of a    28 kDa surface antigen gene family of the tribe Ehrlichieae.    Biochem. Biophys. Res. Commun. 247:636-643.-   Rikihisa, Y., S. A. Ewing, J. C. Fox, A. G. Siregar, F. H. Pasaribu,    and M. B. Malole. 1992. Analyses of Ehrlichia canis and a canine    granulocytic Ehrlichia infection. J. Clin. Microbiol. 30:143-148.-   Singu, V., H. Liu, C. Cheng, and R. R. Ganta. 2005. Ehrlichia    chaffeensis expresses macrophage- and tick cell-specific    28-kilodalton outer membrane proteins. Infect. Immun. 73:79-87.-   Sirigireddy, K. R. and R. R. Ganta. 2005. Multiplex detection of    Ehrlichia and Anaplasma species pathogens in peripheral blood by    real-time reverse transcriptase-polymerase chain reaction. J Mol    Diagn 7:308-316.-   Stockham, S. L., D. A. Schmidt, K. S. Curtis, B. G. Schauf, J. W.    Tyler, and S. T. Simpson. 1992. Evaluation of granulocytic    ehrlichiosis in dogs of Missouri, including serologic status to    Ehrlichia canis, Ehrlichia equi and Borrelia burgdorferi. Am. J.    Vet. Res. 53:63-68.-   Sumner, J. W., J. E. Childs, and C. D. Paddock. 1999. Molecular    cloning and characterization of the Ehrlichia chaffeensis    variable-length PCR target: an antigen-expressing gene that exhibits    interstrain variation. J. Clin. Microbiol. 37:1447-1453.-   Troy, G. C. and S. D. Forrester. 1990. Canine ehrlichiosis, p.    404-418. In C. E. Green (ed.), Infectious diseases of the dog and    cat. W.B. Sauders Co., Philadelphia.-   Unver, A., Y. Rikihisa, R. W. Stich, N. Ohashi, and S. Felek. 2002.    The omp-1 major outer membrane multigene family of Ehrlichia    chaffeensis is differentially expressed in canine and tick hosts.    Infect. Immun. 70:4701-4704.-   Vita et al., 1998, “Novel miniproteins engineered by the transfer of    active sites to small natural scaffolds.” Biopolymers 47:93-100.-   Waner, T., S. Harrus, F. Jongejan, H. Bark, A. Keysary, and A. W.    Cornelissen. 2001. Significance of serological testing for    ehrlichial diseases in dogs with special emphasis on the diagnosis    of canine monocytic ehrlichiosis caused by Ehrlichia canis. Vet.    Parasitol. 95:1-15.-   Weisshoff et al., 1999, “Mimicry of beta II′-turns of proteins in    cyclic pentapeptides with one and without D-amino acids.” Eur. J.    Biochem. 259:776-788.-   Yabsley, M. J., S. E. Little, E. J. Sims, V. G. Dugan, D. E.    Stallknecht, and W. R. Davidson. 2003. Molecular variation in the    variable-length PCR target and 120-kilodalton antigen genes of    Ehrlichia chaffeensis from white-tailed deer (Odocoileus    virginianus). J. Clin. Microbiol. 41:5202-5206.-   Yu, X. J., J. W. McBride, C. M. Diaz, and D. H. Walker. 2000.    Molecular cloning and characterization of the 120-kilodalton protein    gene of Ehrlichia canis and application of the recombinant    120-kilodalton protein for serodiagnosis of canine ehrlichiosis. J.    Clin. Microbiol. 38:369-374.

Yu, X. J., J. W. McBride, C. M. Diaz, and D. H. Walker. 2000. Molecularcloning and characterization of the 120-kilodalton protein gene ofEhrlichia canis and application of the recombinant 120-kilodaltonprotein for serodiagnosis of canine ehrlichiosis. J. Clin. Microbiol.38:369-374.

-   Yu, X. J., J. W. McBride, X. F. Zhang, and D. H. Walker. 2000.    Characterization of the complete transcriptionally active Ehrlichia    chaffeensis 28 kDa outer membrane protein multigene family. Gene    248:59-68.-   Yu, X. J., P. A. Crocquet-Valdes, L. C. Cullman, V. L. Popov,    and D. H. Walker. 1999. Comparison of Ehrlichia chaffeensis    recombinant proteins for serologic diagnosis of human monocytotropic    ehrlichiosis. J. Clin. Microbiol. 37:2568-2575.-   Yu, X. J., P. Crocquet-Valdes, and D. H. Walker. 1997. Cloning and    sequencing of the gene for a 120-kDa immunodominant protein of    Ehrlichia chaffeensis. Gene 184:149-154.-   Yu, X. J., P. Crocquet-Valdes, L. C. Cullman, and D. H.    Walker. 1996. The recombinant 120-kilodalton protein of Ehrlichia    chaffeensis, a potential diagnostic tool. J. Clin. Microbiol.    34:2853-2855.-   Yu, X., J. F. Piesman, J. G. Olson, and D. H. Walker. 1997. Short    report: geographic distribution of different genetic types of    Ehrlichia chaffeensis. Am. J. Trop. Med. Hyg. 56:679-680.-   Zhang, X. F., J. Z. Zhang, S. W. Long, R. P. Ruble, and X. J.    Yu. 2003. Experimental Ehrlichia chaffeensis infection in    beagles. J. Med. Microbiol. 52:1021-1026.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

1. A pharmaceutical composition, comprising one or more of thefollowing: (a) an isolated polypeptide comprising SEQ ID NO:17 or SEQ IDNO:19; (b) an isolated polypeptide that is at least 70% identical to apolypeptide of (a); (c) an isolated polypeptide comprising SEQ ID NO:13;or (d) an isolated polypeptide that is at least 70% identical to SEQ IDNO:13, wherein said polypeptide is dispersed in a pharmaceuticallyacceptable diluent.
 2. The composition of claim 1, wherein (b) isfurther defined as a polypeptide that is at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% identical to a polypeptide of(a).
 3. (canceled)
 4. (canceled)
 5. (canceled)
 6. (canceled)
 7. Thecomposition of claim 1, further defined as a vaccine composition.
 8. Thecomposition of claim 1, wherein the polypeptide is further defined ascomprising one or more carbohydrate moieties.
 9. The composition ofclaim 1, wherein the polypeptide of (a) comprises SEQ ID NO:17 or SEQ IDNO:19.
 10. (canceled)
 11. The composition of claim 1, wherein thepolypeptide of (c) is further defined as being from 24 to 50 amino acidsin length.
 12. The composition of claim 1, wherein the polypeptide of(d) is further defined as being at least 80%, at least 85%, at least90%, or at least 95% identical to SEQ ID NO:13.
 13. (canceled) 14.(canceled)
 15. (canceled)
 16. A pharmaceutical composition comprising anisolated polypeptide encoded by an isolated nucleic acid molecule, saidnucleic acid molecule comprising: (a) a polynucleotide comprising SEQ IDNO:16 or SEQ ID NO:18; or (b) a polynucleotide that is capable ofhybridizing under stringent conditions to the polynucleotide of (a);wherein the polypeptide has at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% identity to SEQ ID NO:17 or SEQID NO:19 and wherein the polypeptide is dispersed in a pharmaceuticallyacceptable diluent.
 17. (canceled)
 18. (canceled)
 19. (canceled) 20.(canceled)
 21. (canceled)
 22. An isolated nucleic acid molecule,comprising: (a) a polynucleotide comprising SEQ ID NO:16 or SEQ IDNO:18; or (b) a polynucleotide that is capable of hybridizing understringent conditions to the polynucleotide of (a) and that encodes apolypeptide having at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% identity to SEQ ID NO:17 or SEQ IDNO:19, wherein said nucleic acid molecule is operably linked to aheterologous promoter.
 23. The isolated nucleic acid molecule of claim22, wherein (a) is further defined as the polynucleotide comprising SEQID NO:16 or as the polynucleotide comprising SEQ ID NO:18. 24.(canceled)
 25. (canceled)
 26. (canceled)
 27. (canceled)
 28. (canceled)29. (canceled)
 30. The isolated nucleic acid molecule of claim 22,wherein the heterologous promoter is a eukaryotic promoter or aprokaryotic promoter.
 31. (canceled)
 32. The isolated nucleic acidmolecule of claim 22, further defined as being comprised in a vector.33. The isolated nucleic acid molecule of claim 32, wherein said vectoris a viral vector or a non-viral vector.
 34. The isolated nucleic acidmolecule of claim 33, wherein said viral vector comprises an adenoviralvector, a retroviral vector, or an adeno-associated viral vector. 35.The isolated nucleic acid molecule of claim 22, comprised in a liposome.36. An isolated antibody that immunologically reacts with one or more ofthe amino acid sequences selected from the group consisting of SEQ IDNO:13, SEQ ID NO:17, and SEQ ID NO:19.
 37. The antibody of claim 36,wherein said antibody is a monoclonal antibody, is comprised inpolyclonal antisera, or is an antibody fragment.
 38. (canceled) 39.(canceled)
 40. (canceled)
 41. (canceled)
 42. A method of inducing animmune response in an individual, comprising the step of delivering tothe individual a therapeutically effective amount of a composition ofclaim
 1. 43. A method of inhibiting E. canis infection in a subject,comprising the step of administering to the subject prior to exposure orsuspected of being exposed to or infected with E. canis, an effectiveamount of a composition of claim
 1. 44. A method of identifying an E.canis infection in an individual, comprising the step of assaying asample from the individual for one or both of the following: (a) apolypeptide of SEQ ID NO:17, SEQ ID NO:19, or both; or (b) an antibodythat immunologically reacts with an amino acid sequence selected fromthe group consisting of SEQ ID NO:13, SEQ ID NO:17, and SEQ ID NO:19.45. (canceled)
 46. (canceled)
 47. (canceled)
 48. (canceled) 49.(canceled)
 50. (canceled)
 51. The method of claim 44, wherein (b) isfurther defined as assaying for an antibody by ELISA.
 52. The method ofclaim 51, wherein the ELISA allows assaying for one or more E. canisantibodies other then the antibody of (b).
 53. The method of claim 52,wherein the other E. canis antibodies are selected from the groupconsisting of antibodies for gp36, gp19, 28/30, and gp200.
 54. A kit,comprising one or more of the following compositions: (a) an isolatedpolypeptide comprising SEQ ID NO:17 or SEQ ID NO:19; (b) an isolatedpolypeptide that is at least 70% identical to a polypeptide of (a); (c)an isolated polypeptide comprising SEQ ID NO:13; (d) an isolatedpolypeptide that is at least 70% identical to SEQ ID NO:13; (e) apolynucleotide comprising SEQ ID NO:16 or SEQ ID NO:18; (f) apolynucleotide that is capable of hybridizing under stringent conditionsto the polynucleotide of (a) and that encodes a polypeptide having atleast 70% identity to SEQ ID NO:17 or SEQ ID NO:19; or (g) an isolatedantibody that immunologically reacts with one or more of the amino acidsequences selected from the group consisting of SEQ ID NO:13, SEQ IDNO:17, and SEQ ID NO:19.
 55. The kit of claim 54, further defined ascomprising two or more of the compositions.
 56. A method of inducing animmune response in an individual, comprising the step of delivering tothe individual a therapeutically effective amount of an antibody ofclaim
 36. 57. A method of inhibiting E. canis infection in a subject,comprising the step of administering to the subject prior to exposure orsuspected of being exposed to or infected with E. canis, an effectiveamount of a composition of claim 36.